Administration of nicotinamide mononucleotide in the treatment of disease

ABSTRACT

Disclosed are methods and compositions related to methods of treating, ameliorating, mitigating, slowing, arresting, preventing or reversing various diseases and conditions, including age-related obesity, age-related increases in blood lipid levels, age-related decreases in insulin sensitivity, age-related decreases in memory function, and age-related changes in eye function such as macular degeneration. The methods comprise administering nicotinamide mononucleotide (NMN) to a subject. In some embodiments, the administration can be oral administration. Also disclosed are pharmaceutical compositions comprising NMN.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims priority to U.S.Non-Provisional application Ser. No. 15/783,845, filed Oct. 13, 2017.application Ser. No. 15/783,845 claims the benefit of and priority toU.S. Non-Provisional application Ser. No. 14/855,293, filed Sep. 15,2015, now U.S. Pat. No. 9,844,561, issued Dec. 19, 2017. applicationSer. No. 14/855,293 claims the benefit of and priority to PCTApplication PCT/US14/30920, filed Mar. 17, 2014. PCT/US14/30920 claimsthe benefit of and priority to U.S. Provisional Patent Application61/801,188 filed Mar. 15, 2013 and U.S. Provisional Patent Application61/947,387 filed Mar. 3, 2014. Each of these applications isincorporated herein by reference, each in its entirety.

GOVERNMENT LICENSE RIGHTS

This invention was made with government support under AG024150 awardedby the National Institutes of Health. The government has certain rightsin the invention.

INTRODUCTION

Age-related obesity is a health-related problem for which new treatmentsand methods of ameliorating, mitigating, or reversing are needed. U.S.Pat. No. 8,268,575 to Imai, S., et al. asserts that “Chemical effectorsfor mammalian NAD biosynthesis can mediate a variety of anti-agingeffects including anti-obesity, neuroprotective, and pancreatic βcell-protective effects as well as be effective to treat cancers.” U.S.Pat. No. 8,017,634 to Sinclair, D. A., et al. teaches treating a cellwith “an agent that increases Nrk enzyme.” U.S. Patent ApplicationPublication 2006/0229265 of Milburn, M., et al. discusses nicotinamideriboside and analogs thereof, including their use in methods of treatingdiseases or conditions, such as diabetes/insulin resistance,hyperlipidemia and obesity. Example 7 of Milburn, M., et al. purports todescribe testing of neuroprotective effects of nicotinamide riboside andnicotinamide mononucleotide on ganglion cell survival. None of thesereferences teach or suggest oral administration of NMN.

Age-related increases in blood lipid levels constitute anotherhealth-related problem for which new treatments and methods ofameliorating, mitigating, or reversing are needed. U.S. Pat. No.7,737,158 of Imai, S., et al. discloses “a process for regulating theconcentration of blood glucose in a mammal, the process comprisingadministering to the mammal a blood glucose concentration-regulatingamount of nicotinamide mononucleotide (NMN) or a salt or prodrugthereof.” This patent is not related to administering NMN for preventingage-associated increases in blood lipids, in particular there is nodisclosure of oral administration of NMN for preventing age-associatedincreases in blood lipids. PCT Patent Application PublicationWO2009062910 of Inufusa, H. states that an objective is to providecompositions effective in reducing blood triglyceride levels and/orcholesterol levels for the treatment and/or prophylaxis ofhypertriglyceridemia, hypercholesterolemia and disease states inducedthereby such as arteriosclerosis and obesity. However, this applicationdoes not disclose administering NMN for controlling age-related bloodlipid increases.

Age-related decreases in insulin sensitivity constitute anotherhealth-related problem for which new treatments and methods ofameliorating, mitigating, or reversing are needed. U.S. Pat. No.7,737,158 to Imai, S., et al. discusses “processes for regulating theconcentration of blood glucose in a mammal. The processes includeadministering to a mammal a blood glucose concentration-regulatingamount of a compound . . . useful in nicotinamide adenine dinucleotide(NAD) biosynthesis. This disclosure does not teach administering NMN,including oral administration of NMN, for improvement of insulinsensitivity in aging.

Age-related loss or decreases in memory function constitute anotherhealth-related problem for which new treatments and methods ofameliorating, mitigating, or reversing are needed. While U.S. PatentApplication Publication 2006/0229265 of Milburn, M., et al. alleges, inExample 7, of “neuroprotective effects” of NMN, no data are presented.This application does not discuss using NMN to treat or prevent memoryloss. U.S. Patent Application Publication 2006/0002914 of Milbrandt, J.,et al. discloses administering an agent that acts by increasing NADactivity in diseased and/or injured neurons in the treatment of diseasessuch as Alzheimer's. There is no explicit teaching of administering NMNto improve memory function under normal aging conditions.

Age-related loss or decreases in eye function constitute anotherhealth-related problem for which new treatments and methods ofameliorating, mitigating, or reversing are needed. U.S. PatentApplication Publication 2007/0014833 of Milburn, M., et al. disclosesuse of“sirtuin modulators” to treat vision impairment. U.S. PatentApplication Publication 2006/0229265 of Milburn, M., et al. states“Conditions of the eye can be treated or prevented by, e.g., systemic,topical, intraocular injection of a sirtuin modulating compound, or byinsertion of a sustained release device that releases asirtuin-modulating compound. Neither of these publications discloseadministration of NMN for the prevention of decline of eye functionduring aging.

Age-related dry eye constitutes another health-related problem for whichnew treatments and methods of ameliorating, mitigating, or reversing areneeded. Dry eye is one of the most prevalent eye disorders, particularlyamong the elderly, and no fundamental treatments are yet available(Tsubota, K., et al. Cornea. 31 Suppl 1, S1-S8, 2012). Decrease inlacrimal gland secretory function might be a possible cause ofage-associated dry eye diseases (Kawashima, M., et al. Biochem Biophys.Res. Commun. 397, 724-728, 2010).

Age-related cognitive impairment constitutes another health-relatedproblem for which new treatments and methods of ameliorating,mitigating, or reversing are needed. Aging is a negative regulator ofadult neural stem and progenitor cell (NSPC) proliferation (Artegiani,B., et al.) While NSPC proliferation declines exponentially throughoutlife (Artegiani & Calegari, 2012), quiescent NSPCs can be reactivated inthe aged murine hippocampus by multiple environmental stimuli (Decker etal, 2002; Jin et al, 2003; Lugert et al, 2010). Aging can reduce levelsof the essential cofactor nicotinamide adenine dinucleotide (NAD+) inmultiple peripheral tissues (Yoshino et al, 2011).

U.S. patent application Ser. No. 12/524,718 Milbrandt, J., et al.discloses experimental applications of NMN. This reference does notmention administering NMN to affect NSPCs or for oligodendrocyteproliferation.

Sasaki, Y., et al., J. Neurosci. 26, 8484-8491, 2006 disclosesapplications of NMN. This article does not disclose administration ofNMN to affect NSPCs or promote proliferation of oligodendrocytes. U.S.Application US20130059384 A1 of Tilly, J. L., et al. discloses the useof NMN for enhancing female fertility.

Photoreceptor neuron dysfunction and cell death is the leading cause ofblindness over the lifespan in humans. Photoreceptor neuron dysfunctionconstitutes another health-related problem for which new treatments andmethods of ameliorating, mitigating, or reversing are needed.

PCT Application PCT/US2006/011930 (Pub No. WO2006105403) of Dipp, M., etal. disclosed methods of treating vision impairment by administration ofa sirtuin modulator. In example 10 of this reference, the investigatorstested neuroprotective effects of nicotinamide mononucleotide (NMN) in aretinal ganglion cell injury model. However, the retinal ganglion cellsdisclosed in this application not classical photoreceptors (rods andcones).

U.S. Patent Application Publication US20100047177 of Milbrandt, J., etal. discusses administering to a mammal an agent that increases NADactivity in diseased and/or injured neurons or supporting cells. Theapplication does not specify protection or treatment of rods and coneswith NMN.

U.S. Pat. No. 7,776,326 of Milbrandt, J., et al. discusses methods oftreating or preventing axonal degradation in neuropathic diseases inmammals by administering an agent that can increase sirtuin activity indiseased/injured neuronal cells. The patent does not specify protectionor treatment of rods and cones with NMN. This patent does not discloseadministration of NMN to maintain NSPCs or promote oligodendrocyteproliferation.

“Stimulation of nicotinamide adenine dinucleotide biosynthetic pathwaysdelays axonal degeneration after axotomy” J. Neurosci. 26, 8484-8491,2006 of Sasaki, Y., et al. discusses adding NMN to cultured neurons.This article does not disclose administration of NMN to retinal cells invivo.

Chinese Patent CN 101601679 B “Application of nicotinamidemononucleotide” discloses applications of NMN for prevention of stroke.

PCT Application PCT/IB2012/001146 of Alvarez, C. C., et al. disclosestreating a mitochondrial dysfunction with a compound that increasesintracellular nicotinamide adenine dinucleotide (NAD+) in an amountsufficient to activate SIRT1 or SIRT3. The application does not discloseadministration of NMN to neuronal cells. This reference does not mentionNSPCs.

U.S. Pat. No. 7,737,158 of Imai, S., et al. discloses processes forregulating blood glucose concentration by administration of NMN. Thepatent does not teach administration of NMN to rod/cone-typephotoreceptor neurons. This reference does not discuss administration ofNMN to treat NSPCs or for age-related diseases or neurodegenerativediseases unrelated to glucose levels.

Revollo, J. R., et al., Cell Metab. 6, 363-375, 2007; Ramsey, K. M., etal., Aging Cell 7, 78-88, 2008; and Yoshino, J., et al., Cell Metab. 14,528-536, 2011 do not disclose administration of NMN for treatingphotoreceptor degeneration, retinal degeneration, or maculardegeneration. Exp. Eye Res. 108, 76-83, 2013 of Bai, S., et al. does notdisclose targeting rod/cone-type photoreceptor neurons with NMN. Thesearticles do not discuss NSPCs.

SUMMARY

The present inventors describe administration of nicotinamidemononucleotide (NMN), as illustrated in (FIG. 1), to a subject such as avertebrate, including a human or other mammal, a bird, a reptile, a fishor other aquatic organism.

In addition, also disclosed in various embodiments are compositionscomprising NMN. In various configurations, these compositions canfurther comprise one or more excipients. These compositions can be usedfor administration of NMN for the treatment, amelioration, mitigation,slowing, arrest, prevention and/or reversal of age-associateddegenerative changes.

In some embodiments, any of the dosages of the present teachings ofnicotinamide mononucleotide (NMN), a salt thereof and/or a prodrugthereof can be used to treat any of the diseases of the presentteachings.

In various embodiments, the present teachings include a pharmaceuticallyacceptable composition comprising, consisting essentially of, orconsisting of nicotinamide mononucleotide (NMN), a salt thereof and/or aprodrug thereof and at least one pharmaceutically acceptable excipient.In various configurations, a pharmaceutically acceptable compositioncan, comprise, consist essentially of, or consist of a single dosageformulation. In some configurations, a single dosage formulation can bea sustained-release formulation.

In various configurations, a pharmaceutically acceptable composition ofthe present teachings can be a formulation including a pill, a tablet, acaplet, a capsule, a chewable tablet, a quick dissolve tablet, a powder,an effervescent tablet, a hard gelatin capsule, a soft gelatin capsule,a non-aqueous liquid, an aqueous liquid, a granule, a capsule, asuspension, a solution, an emulsion, a syrup, a sterilized aqueoussuspension or solution, a non-aqueous suspension or solution, alyophilized formulation, or a suppository.

In some embodiments, a pharmaceutically acceptable composition of thepresent teachings can be a single dosage formulation. In variousembodiments, a single dosage formulation can comprise an entericcoating. In some embodiments, a pharmaceutically acceptable compositionof the present teachings can comprise, consist essentially of, orconsist of NMN, a salt thereof and/or a prodrug thereof in an amount ofabout 100 mg, from 100 mg to 2000 mg, about 2000 mg, or greater. In someembodiments, a pharmaceutically acceptable composition of the presentteachings can comprise, consist essentially of, or consist of NMN, asalt thereof and/or a prodrug thereof in an amount of about 100 mg,about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg,about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg,about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600mg, about 1700 mg, about 1800 mg, about 1900 mg, about 2000 mg, orgreater. In some embodiments, a pharmaceutically acceptable compositionof the present teachings can comprise, consist essentially of, orconsist of NMN, a salt thereof and/or a prodrug thereof in an amount of50-150 mg, 151-250 mg, 251-350 mg, 351-450 mg, 451-550 mg, 561-650 mg,651-750 mg, 751-860 mg, 861-950 mg, 951-1050 mg, 1051-1150 mg, 1151-1250mg, 1251-1350 mg, 1351-1450 mg, 1451-1550 mg, 1551-1650 mg, 1651-1750mg, 1751-1850 mg, 1851-1950 mg, 1951-2000 mg, or greater. In someembodiments, a pharmaceutically acceptable composition of the presentteachings can comprise, consist essentially of, or consist of NMN, asalt thereof and/or a prodrug thereof in an amount of at least 0.5 mg upto about 6800 mg, such as, without limitation, 0.5 mg, about 0.5 mg, 1mg, about 1 mg, 5 mg, about 5 mg, 10 mg, about 10 mg, 20 mg, about 20mg, 30 mg, about 30 mg, 40 mg, about 40 mg, 50 mg, about 50 mg, 60 mg,about 60 mg, 70 mg, about 70 mg, 80 mg, about 80 mg, 90 mg, about 90 mg,100 mg, about 100 mg, 150 mg, about 150 mg, 200 mg, about 200 mg, 250mg, about 250 mg, 300 mg, about 300 mg, 400 mg, about 400 mg, 450 mg,about 450 mg, 500 mg, about 500 mg, 600 mg, about 600 mg, 680 mg, about680 mg, 700 mg, about 700 mg, 800 mg, about 800 mg, 900 mg, about 900mg, 1000 mg, about 1000 mg, 1100 mg, about 1100 mg, 1130 mg, about 1130mg, 1200 mg, about 1200 mg, 1300 mg, about 1300 mg, 1350 mg, about 1350mg, 1360 mg, about 1360 mg, 1400 mg, about 1400 mg, 1500 mg, about 1500mg, 1600 mg, about 1600 mg, 1700 mg, about 1700 mg, 1800 mg, about 1800mg, 1900 mg, about 1900 mg, 2000 mg, about 2000 mg, 2040 mg, about 2040mg, 2100 mg, about 2100 mg, 2200 mg, about 2200 mg, 2250 mg, about 2250mg, 2300 mg, about 2300 mg, 2400 mg, about 2400 mg, 2500 mg, about 2500mg, 2600 mg, about 2600 mg, 2700 mg, about 2700 mg, 2800 mg, about 2800mg, 2900 mg, about 2900 mg, 3000 mg, about 3000 mg, 3100 mg, about 3100mg, 3200 mg, about 3200 mg, 3300 mg, about 3300 mg, 3400 mg, about 3400mg, 3500 mg, about 3500 mg, 3600 mg, about 3600 mg, 4000 mg, about 4000mg, 4500 mg, about 4500 mg, 5000 mg, about 5000 mg, 5500 mg, about 5500mg, 6000 mg, about 6000 mg, 6500 mg, about 6500 mg, 6800 mg, or about6800 mg.

In some embodiments, a pharmaceutically acceptable composition of thepresent teachings can comprise a food product. In some embodiments, apharmaceutically acceptable composition of the present teachings cancomprise a composition suitable for oral administration, sublingualadministration, parenteral administration, administration by injection,subcutaneous injection, intramuscular injection, intraperitonealinjection, intra-ocular injection, direct ocular application (eye drop)or a combination thereof.

In some embodiments, a pharmaceutically acceptable composition of thepresent teachings can further comprise least one excipient. In someembodiments, the at least one excipient can comprise a bulking agent, atableting agent, a dissolution agent, a wetting agent, a lubricant, acoloring, a flavoring, a disintegrant, a coating, a binder, anantioxidant, a taste masking agent, a sweetener, or a combinationthereof. In some embodiments, a bulking agent can comprise mannitol,sorbitol, sucrose, trehalose, or a combination thereof.

In some embodiment, a pharmaceutically acceptable composition of thepresent teachings can be formulated as an orally disintegrating capsule,tablet, pill or wafer. In some embodiment, a pharmaceutically acceptablecomposition of the present teachings can be formulated as a liquid,syrup, or spray.

In some embodiment, a pharmaceutically acceptable composition of thepresent teachings can further comprise at least one vitamin or nutrient.In some embodiments, a vitamin or nutrient can be vitamin C, vitamin D3,vitamin E, vitamin B1, vitamin B2, niacin, vitamin B6, folic acid,vitamin B12, pantothenic acid, biotin, magnesium, zinc, copper,selenium, chromium, alpha lipoic acid, b co-enzyme Q-10, lutein,lycopene, or a combination thereof. In some embodiments, a vitamin ornutrient can be an amount comprising 150 mg to about 750 mg of vitaminC, from about 315 IUs to about 1800 IUs of vitamin D3, from about 75 IUsto about 150 IUs of vitamin E, from about 15 mg to about 35 mg ofvitamin B1, from about 1.7 mg to about 5.1 mg of vitamin B2, from about20 mg to about 50 mg of niacin, from about 20 mg to about 50 mg ofvitamin B6, from about 0.5 mg to about 2.5 mg of folic acid, from about35 mcg to about 105 mcg of vitamin B12, from about 2.5 mg to about 7.5mg of pantothenic acid, from about 50 mcg to about 450 mcg of biotin,from about 15 mg to about 55 mg of magnesium, from about 15 mg to about55 mg of zinc, from about 0.5 to about 1.5 mg of copper, from about 75mcg to about 175 mcg of selenium, from about 75 mcg to about 225 mcg ofchromium, from about 10 mg to about 40 mg of alpha lipoic acid, fromabout 20 mg to about 50 mg of co-enzyme Q-10, from about 350 mcg toabout 3 mg of lutein, from about 100 mcg to about 750 mcg of lycopene,or a combination thereof.

In some embodiments, a vitamin or nutrient can be about 500 mg ofascorbic acid, about 400 IUs of cholecalciferol, about 125 IUs ofd-alpha tocopherol succinate, about 35 mg of thiamine mononitrate, about5.1 mg of riboflavin, about 50 mg of niacinamide, about 50 mg ofpyridoxine HCl, about 2.5 mg of folic acid, about 105 mcg ofcyanocobalamin, about 7.5 mg of d-calcium pantothenate, about 75 mcg ofd-biotin, about 55 mg of dimagnesium malate, about 55 mg of zincbisglycinate chelate, about 1.5 mg of copper amino acid chelate, about175 mcg of selenium amino acid chelate, about 225 mcg of chromium aminoacid chelate, about 10 mg of alpha lipoic acid, about 50 mg of co-enzymeQ-10, about 400 mcg of lutein, about 125 mcg of lycopene, or acombination thereof.

In some embodiments, an excipient of the present teachings can bedicalcium phosphate, microcrystalline cellulose, stearic acid,croscarmellose sodium, magnesium trisilicate, magnesium stearate,hydroxypropyl methylcellulose, hypromellose, titanium dioxide,tripotassium citrate, polyvinyl alcohol, fumed silica, citric acid,polyethylene glycol, talc, or a combination thereof. In someembodiments, an excipient of the present teachings can be about 100 mgto about 300 mg of dicalcium phosphate, from about 25 mg to about 75 mgof microcrystalline cellulose, from about 10 mg to about 30 mg ofstearic acid, from about 10 mg to about 30 mg of croscarmellose sodium,from about 5 mg to about 15 mg of magnesium trisilicate, from about 5 mgto about 15 mg of magnesium stearate, from about 5 mg to about 15 mg ofhydroxypropyl methylcellulose, or a combination thereof.

In some embodiments, an excipient of the present teachings can be about200 mg of dicalcium phosphate, about 50 mg of microcrystallinecellulose, about 20 mg of stearic acid, about 20 mg of croscarmellosesodium, about 10 mg of magnesium trisilicate, about 10 mg of magnesiumstearate, about 10 ng of hydroxypropyl methylcellulose, or a combinationthereof.

In some embodiments, a pharmaceutically acceptable composition of thepresent teachings can be a sustained release formulation of nicotinamidemononucleotide for oral administration. In some embodiments, a sustainedrelease formulation of nicotinamide mononucleotide for oraladministration can comprise nicotinamide mononucleotide as an activeingredient that is released from the formulation along a pre-determinedrelease profile, wherein the formulation comprises an extended releasecomponent and an immediate release component, wherein the extendedrelease component is contained in at least one population of beads andreleases nicotinamide mononucleotide in a continuous manner and eachbead population is coated with its own release controlling coating andcharacterized by its own rate of release.

In some embodiments, an extended release component can release thenicotinamide mononucleotide in vivo in a continuous manner. In someembodiments, an extended release component can release the nicotinamidemononucleotide in vivo in a continuous manner and 80% of thenicotinamide mononucleotide can be released in vivo in a period of timeselected from not more than 24 hours, not more than 16 hours, not morethan 12 hours, not more than 8 hours or not more than 4 hours. In someembodiments, an immediate release component of the present teachings canbe an enhanced immediate release (EIR) composition comprising acomplexing agent, an enhancing agent, or a combination. In someembodiments, an EIR composition can exhibit an in vitro release profilesuch that 80% of the active ingredient is dissolved in not more than 30min. In some embodiments, an EIR composition can exhibit an in vitrorelease profile selected from a group consisting of: a) a dissolution ofat least 50% of the active compound in not more than 10 minutes, b) adissolution of at least 70% of the active compound in not more than 10minutes, c) a dissolution of at least 25% of the active compound in notmore than 5 minutes, d) a dissolution of at least 40% of the activecompound in not more than 5 minutes, or e) a dissolution of at least 55%of the active compound in not more than 5 minutes.

In some embodiments, a pharmaceutically acceptable composition of thepresent teachings can include a complexing agent. In some embodiments, acomplexing agent can be hydroxypropyl-beta-cyclodextrin,beta-cyclodextrin, gamma-cyclodextrin, alpha-cyclodextrin, derivativesthereof, or a combination thereof.

In some embodiments, a pharmaceutically acceptable composition of thepresent teachings can include an enhancing agent. In some embodiments,an enhancing agent can be a solubility enhancing agent, a dissolutionenhancing agent, an absorption enhancing agent, a penetration enhancingagent, a surface active agent, a stabilizer, an enzyme inhibitor, ap-glycoprotein inhibitor, a multidrug resistance protein inhibitor, or acombination thereof.

In some embodiments, an enhancing agent can be Vitamin E TPGS, glutamicacid, glycine, sorbitol, mannose, amylose, maltose, mannitol, lactose,sucrose, glucose, xylitose, dextrins, glycerolpolyethylene glycoloxystearate, PEG-32 glyceryl palmitostearate, sodium lauryl sulfate,polyoxyethylene sorbitan monooleate, benzyl alcohol, sorbitanmonolaurate, Poloxamer 407, PEG3350, PVP K25, oleic acid, glycerylmonooleate, sodium benzoate, cetyl alcohol, sucrose stearate,crospovidone, sodium starch glycolate, croscarmellose sodium,carboxymethylcellulose, starch, pregelatinized starch, HPMC, substitutedhydroxypropylcellulose, microcrystalline cellulose sodium bicarbonate,calcium citrate, sodium docusate, menthol, or any combination thereof.

In some embodiments, a pharmaceutically acceptable composition of thepresent teachings can include at least a part of the active ingredientin a form of micronized particles. In some embodiments, a micronizedparticle can have an average size of from about 2 μm to about 100 μm.

In some embodiments, a pharmaceutically acceptable composition of thepresent teachings can include a specific amount of each componentdetermined according to the purpose of administration and thepre-determined release profile, and the total amount of NMN in theformulation is from 0.5 to 3000 mg.

In some embodiments, a population of beads of the present teachings cancomprise an inert carrier, NMN, an optional enhancer, a releasecontrolling coating that comprises a coating material, a pore former, anexcipient, or a combination thereof. In some embodiments, an inertcarrier of the present teachings can be a cellulose sphere, silicondioxide, starch, a sugar sphere, or a combination thereof.

In some embodiments, an enhancer can be a solubility enhancer, adissolution enhancer, a permeability enhancer, a stabilizer, acomplexing agent, an enzyme inhibitor, a p-glycoprotein inhibitor, amultidrug resistance protein inhibitor, or a combination thereof.

In some embodiments, a coating material of the present teachings can beethylcellulose, methylcellulose, hydroxypropyl cellulose,hydroxypropylmethyl cellulose, cellulose acetate, cellulose acetatephthalate, polyvinyl alcohol, polyacrylates, polymethacrylates andcopolymers thereof; and/or a pore former is selected from a groupconsisting of glucose, fructose, mannitol, mannose, galactose, sorbitol,pullulan, dextran, water-soluble hydrophilic polymers,hydroxyalkylcelluloses, carboxyalkylcelluloses,hydroxypropylmethylcellulose, cellulose ethers, acrylic resins,polyvinylpyrrolidone, cross-linked polyvinylpyrrolidone, polyethyleneoxide, Carbowaxes, Carbopol, diols, polyols, polyhydric alcohols,polyalkylene glycols, polyethylene glycols, polypropylene glycols orblock polymers thereof, polyglycols, poly(a-w)alkylenediols; inorganiccompounds selected from a group consisting of alkali metal salts andalkaline earth metal salts, or combinations thereof.

In some embodiments, an amount of an individual bead population isdetermined according to a pre-determined release profile. In someembodiments, a pre-determined release profile can comprise a sustainedrate of release after an initial immediate release. IN some embodiments,a pharmaceutically acceptable composition of the present teachings canbe suitable for once a day oral administration. In some embodiments, apopulation of beads can comprise, consist essentially of, or consist ofextended release NMN beads additionally comprising an immediate releasecomponent coated on top of the release controlling coating. In someembodiments, a formulation of a pharmaceutically acceptable compositionof the present teachings can comprise an enhancer contained in a layerseparate from the release controlling coating. In some embodiments, aformulation of a pharmaceutically acceptable composition of the presentteachings can comprise at least one enhancing agent wherein theenhancing agent is incorporated into the formulation in the form of apowder or of a population of beads that are optionally characterized bya controlled rate of release, and wherein the enhancing agent isseparated from the active ingredient.

In various configurations, a composition of the present teachings cancomprise nicotinamide mononucleotide (NMN), a pharmaceutical salt ofNMN, or a prodrug of NMN. In various configurations, a salt can be apharmaceutically acceptable salt; that is, a salt prepared frompharmaceutically acceptable non-toxic acids, including inorganic acidsand organic acids. Non-limiting examples of suitable non-toxic acidsinclude inorganic and organic acids of basic residues such as amines,for example, acetic, benzenesulfonic, benzoic, amphorsulfonic, citric,ethenesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric,isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic,nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, barbaricacid, p-toluenesulfonic and the like; and alkali or organic salts ofacidic residues such as carboxylic acids, for example, alkali andalkaline earth metal salts derived from the following bases: sodiumhydride, sodium hydroxide, potassium hydroxide, calcium hydroxide,aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinchydroxide, ammonia, trimethylammonia, triethylammonia, ethylenediamine,lysine, arginine, ornithine, choline, N,N″-dibenzylethylenediamine,chloroprocaine, diethanolamine, procaine, n-benzylphenethylamine,diethylamine, piperazine, tris(hydroxymethyl)-aminomethane,tetramethylammonium hydroxide, and the like. Pharmaceutically acceptablesalts can be prepared by reacting the free acid or base forms of thesecompounds with a stoichiometric amount of the appropriate base or acidin water or in an organic solvent, or in a mixture of the two;generally, nonaqueous media like ether, ethyl acetate, ethanol,isopropanol, or acetonitrile are preferred. Lists of suitable salts canbe found in Remington's Pharmaceutical Sciences, 17th ed., MackPublishing Company, Easton, Pa., 1985, p. 1418, the disclosure of whichis hereby incorporated by reference.

NMN can be delivered in prodrug form. Thus, the present teachings areintended to cover prodrugs of NMN, methods of delivering the same andcompositions containing them. A “prodrug” can include any covalentlybonded carriers which release an active drug in vivo when such prodrugis administered to a mammalian subject. In various configurations, aprodrug can be prepared by modifying functional groups present in thecompound in such a way that the modifications are cleaved, either inroutine manipulation or in vivo, to the parent compound. In variousconfigurations, prodrugs include, without limitation, compounds of thepresent teachings wherein a hydroxyl or amino group can be bonded to anygroup that, when the prodrug is administered to a mammalian subject,cleaves to form a free hydroxyl or free amino group, respectively.Non-limiting examples of prodrugs include acetate, formate, and benzoatederivatives of alcohol and amine functional groups in the compounds andconjugates of the present teachings. Prodrugs of NMN can be, within thescope of sound medical judgment, suitable for use in contact with thetissues of humans and lower animals without undue toxicity, irritation,allergic response, and the like, commensurate with a reasonablebenefit/risk ratio, and effective for their intended use, as well as thezwitterionic forms, where possible, of the compounds of the presentteachings. In some configurations, prodrugs can refer to compounds thatcan be transformed in vivo to yield NMN, for example by hydrolysis inblood.

In some embodiments, NMN can be dispersed in a pharmaceuticallyacceptable carrier prior to administration to a subject. In variousembodiments, a carrier, also known in the art as an excipient, vehicle,auxiliary, adjuvant, or diluent, can be a substance that ispharmaceutically inert, can confer a suitable consistency or form to thecomposition, and does not diminish the efficacy of the NMN. A carriercan be considered to be “pharmaceutically or pharmacologicallyacceptable” if it does not lead to pharmaceutically unacceptableadverse, allergic or other untoward reactions when administered to asubject, including a mammalian subject.

The selection of a pharmaceutically acceptable carrier can also, inpart, be a function of the route of administration. For example,suitable routes of administration include, but are not limited to, oral,parenteral (e.g., intravenous, intraarterial, subcutaneous, rectal,subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal,intraperitoneal, or intrasternal), topical (nasal, transdermal, ocularsuch as eyedrops, intraocular), intravesical, intrathecal, enteral,pulmonary, intralymphatic, intracavital, vaginal, transurethral,intradermal, aural, intramammary, buccal, orthotopic, intratracheal,intralesional, percutaneous, endoscopical, transmucosal, sublingual andintestinal administration.

Pharmaceutically acceptable carriers for use in the present teachingscan be selected based upon a number of factors: the particular compoundused, and its concentration, stability and intended bioavailability; thesubject, its age, size and general condition; and the route ofadministration. Suitable nonaqueous, pharmaceutically-acceptable polarsolvents include, but are not limited to, alcohols (e.g., α-glycerolformal, β-glycerol formal, 1,3-butyleneglycol, aliphatic or aromaticalcohols having 2-30 carbon atoms such as methanol, ethanol, propanol,isopropanol, butanol, t-butanol, hexanol, octanol, amylene hydrate,benzyl alcohol, glycerin (glycerol), glycol, hexylene glycol,tetrahydrofurfuryl alcohol, lauryl alcohol, cetyl alcohol, or stearylalcohol, fatty acid esters of fatty alcohols such as polyalkyleneglycols (e.g., polypropylene glycol, polyethylene glycol), sorbitan,sucrose and cholesterol); amides (e.g., dimethylacetamide (DMA), benzylbenzoate DMA, dimethylformamide, N-(β-hydroxyethyl)-lactamide,N,N-dimethylacetamide amides, 2-pyrrolidinone, 1-methyl-2-pyrrolidinone,or polyvinylpyrrolidone); esters (e.g., 1-methyl-2-pyrrolidinone,2-pyrrolidinone, acetate esters such as monoacetin, diacetin, andtriacetin, aliphatic or aromatic esters such as ethyl caprylate oroctanoate, alkyl oleate, benzyl benzoate, benzyl acetate,dimethylsulfoxide (DMSO), esters of glycerin such as mono, di, ortri-glyceryl citrates or tartrates, ethyl benzoate, ethyl acetate, ethylcarbonate, ethyl lactate, ethyl oleate, fatty acid esters of sorbitan,fatty acid derived PEG esters, glyceryl monostearate, glyceride esterssuch as mono, di, or tri-glycerides, fatty acid esters such as isopropylmyristrate, fatty acid derived PEG esters such as PEG-hydroxyoleate andPEG-hydroxystearate, N-methyl pyrrolidinone, pluronic 60,polyoxyethylene sorbitol oleic polyesters such as poly(ethoxylated)30-60 sorbitol poly(oleate)2-4, poly(oxyethylene)15-20 monooleate,poly(oxyethylene)15-20 mono 12-hydroxystearate, andpoly(oxyethylene)15-20 mono ricinoleate, polyoxyethylene sorbitan esterssuch as polyoxyethylene-sorbitan monooleate, polyoxyethylene-sorbitanmonopalmitate, polyoxyethylene-sorbitan monolaurate,polyoxyethylene-sorbitan monostearate, and Polysorbate® 20, 40, 60 or 80from ICI Americas, Wilmington, Del., polyvinylpyrrolidone, alkyleneoxymodified fatty acid esters such as polyoxyl 40 hydrogenated castor oiland polyoxyethylated castor oils (e.g., Cremophor® EL solution orCremophor® RH 40 solution), saccharide fatty acid esters (i.e., thecondensation product of a monosaccharide (e.g., pentoses such as ribose,ribulose, arabinose, xylose, lyxose and xylulose, hexoses such asglucose, fructose, galactose, mannose and sorbose, trioses, tetroses,heptoses, and octoses), disaccharide (e.g., sucrose, maltose, lactoseand trehalose) or oligosaccharide or mixture thereof with a C4-C22 fattyacid(s) (e.g., saturated fatty acids such as caprylic acid, capric acid,lauric acid, myristic acid, palmitic acid and stearic acid, andunsaturated fatty acids such as palmitoleic acid, oleic acid, elaidicacid, erucic acid and linoleic acid)), or steroidal esters); alkyl,aryl, or cyclic ethers having 2-30 carbon atoms (e.g., diethyl ether,tetrahydrofuran, dimethyl isosorbide, diethylene glycol monoethylether); glycofurol (tetrahydrofurfuryl alcohol polyethylene glycolether), ketones having 3-30 carbon atoms (e.g., acetone, methyl ethylketone, methyl isobutyl ketone); aliphatic, cycloaliphatic or aromatichydrocarbons having 4-30 carbon atoms (e.g., benzene, cyclohexane,dichloromethane, dioxolanes, hexane, n-decane, n-dodecane, n-hexane,sulfolane, tetramethylenesulfon, tetramethylenesulfoxide, toluene,dimethylsulfoxide (DMSO), or tetramethylenesulfoxide); oils of mineral,vegetable, animal, essential or synthetic origin (e.g., mineral oilssuch as aliphatic or wax-based hydrocarbons, aromatic hydrocarbons,mixed aliphatic and aromatic based hydrocarbons, and refined paraffinoil, vegetable oils such as linseed, tung, safflower, soybean, castor,cottonseed, groundnut, rapeseed, coconut, palm, olive, corn, corn germ,sesame, persic and peanut oil and glycerides such as mono-, di- ortriglycerides, animal oils such as fish, marine, sperm, cod-liver,haliver, squalene, squalane, and shark liver oil, oleic oils, andpolyoxyethylated castor oil); alkyl or aryl halides having 1-30 carbonatoms and optionally more than one halogen substituent; methylenechloride; monoethanolamine; petroleum benzin; trolamine; omega-3polyunsaturated fatty acids (e.g., alpha-linolenic acid,eicosapentaenoic acid, docosapentaenoic acid, or docosahexaenoic acid);polyglycol ester of 12-hydroxystearic acid and polyethylene glycol(Solutol) HS-15, from BASF, Ludwigshafen, Germany); polyoxyethyleneglycerol; sodium laurate; sodium oleate; or sorbitan monooleate.

Other pharmaceutically acceptable solvents for use in the presentteachings are well known to those of ordinary skill in the art, and areidentified in The Chemotherapy Source Book (Williams & WilkensPublishing), The Handbook of Pharmaceutical Excipients, (AmericanPharmaceutical Association, Washington, D.C., and The PharmaceuticalSociety of Great Britain, London, England, 1968), Modern Pharmaceutics,(Banker, G., et al., eds., 3d ed.) (Marcel Dekker, Inc., New York, N.Y.,1995), The Pharmacological Basis of Therapeutics, (Goodman & Gilman,McGraw Hill Publishing), Pharmaceutical Dosage Forms, (H. Lieberman etal., eds.) (Marcel Dekker, Inc., New York, N.Y., 1980), Remington'sPharmaceutical Sciences (A. Gennaro, ed., 19th ed.) (Mack Publishing,Easton, Pa., 1995), The United States Pharmacopeia 24, The NationalFormulary 19, (National Publishing, Philadelphia, Pa., 2000), A. J.Spiegel et al., and Use of Nonaqueous Solvents in Parenteral Products,J. Pharm. Sci. 52, 917-927, 1963.

In some embodiments, the present teachings include methods of treating,ameliorating, mitigating, slowing, arresting, preventing or reversingage-associated obesity in a subject. In some embodiments, the presentteachings include methods of treating, ameliorating, mitigating,slowing, arresting, preventing or reversing age-associated increases inblood lipid levels in a subject. In some embodiments, the presentteachings include methods of treating, ameliorating, mitigating,slowing, arresting, preventing or reversing age-associated loss ofinsulin sensitivity in a subject. In some embodiments, the presentteachings include methods of treating, ameliorating, mitigating,slowing, arresting, preventing or reversing age-associated impairment ofmemory function in a subject. In some embodiments, the present teachingsinclude methods of treating, ameliorating, mitigating, slowing,arresting, preventing or reversing age-associated decline in eyefunction in a subject. In some embodiments, the present teachingsinclude methods of treating, ameliorating, mitigating, slowing,arresting, preventing or reversing age-associated retinal degenerationin a subject. In some embodiments, the present teachings include methodsof treating, ameliorating, mitigating, slowing, arresting, preventing orreversing dry eye. In some embodiments, the present teachings includemethods of treating, ameliorating, mitigating, slowing, arresting,preventing or reversing age-associated dry eye.

In various embodiments, these methods can each independently comprise,consist essentially of, or consist of administering to a subject apharmaceutically effective amount of nicotinamide mononucleotide (NMN).In some embodiments, NMN can be administered at a dosage rate of about100 mg per day, from 100 mg per day to 2000 mg per day, or about 2000 mgper day. In some embodiments, NMN can be administered at a dosage rateof 0.5 mg, about 0.5 mg, 1 mg, about 1 mg, 5 mg, about 5 mg, 10 mg,about 10 mg, 20 mg, about 20 mg, 30 mg, about 30 mg, 40 mg, about 40 mg,50 mg, about 50 mg, 60 mg, about 60 mg, 70 mg, about 70 mg, 80 mg, about80 mg, 90 mg, about 90 mg, about 100 mg per day, 100 mg per day, 150 mg,about 150 mg, about 200 mg per day, 200 mg per day, about 300 mg perday, 300 mg per day, about 400 mg per day, 400 mg per day, about 500 mgper day, 500 mg per day, about 600 mg per day, 600 mg per day, about 700mg per day, 700 mg per day, about 800 mg per day, 800 mg per day, about900 mg per day, 900 mg per day, about 1000 mg per day, 1000 mg per day,about 1100 mg per day, 1100 mg per day, about 1200 mg per day, 1200 mgper day, about 1300 mg per day, 1300 mg per day, 1350 mg, about 1350 mg,about 1400 mg per day, 1400 mg per day, about 1500 mg per day, 1500 mgper day, about 1600 mg per day, 1600 mg per day, about 1700 mg per day,1700 mg per day, about 1800 mg per day, 1800 mg per day, about 1900 mgper day, 1900 mg per day, about 2000 mg per day, 2000 mg per day, 2040mg, about 2040 mg, 2250 mg, about 2250 mg, 2260 mg, about 2260 mg, 2700mg, about 2700 mg, 2720 mg, about 2720 mg, 3400 mg, about 3400 mg, 3390mg, about 3390 mg, 3400 mg, about 3400 mg, 3600 mg, about 3600 mg, 4080mg, about 4080 mg, 4500 mg, about 4500 mg, 4520 mg, about 4520 mg, 5440mg, about 5440 mg, 5650 mg, about 5650 mg, 6800 mg, about 6800 mg, or analternation or combination thereof. In some embodiments, NMN can beadministered at a dosage rate of about 100 mg/kg body weight/day, from100 mg/kg body weight/day to 500 mg/kg body weight/day, or about 500mg/kg body weight/day. In some embodiments, NMN can be administered at adosage rate of about 100 mg/kg body weight/day, from 100 mg/kg bodyweight/day to 300 mg/kg body weight/day, or about 300 mg/kg bodyweight/day. In some embodiments, these methods can compriseadministering to a subject any of the pharmaceutically acceptablecompositions of the present teachings. In some embodiments, thesemethods can comprise, consist essentially of or consist of administeringa formulation once per day. In some embodiments, these methods cancomprise, consist essentially of or consist of administering aformulation twice per day.

In some embodiments, the present teachings include methods of increasingNAD+ levels in a subject through administration of NMN. In someembodiments, the present teachings include methods of treatingage-associated defects in neural stem/progenitor cell (NSPC)functionality in a subject through administration of NMN. In someembodiments, the present teachings include methods of reducingage-associated decrease in a NSPC population in a subject throughadministration of NMN. In some embodiments, the present teachingsinclude methods of maintaining at least one NSPC in a subject throughadministration of NMN. In some embodiments, the present teachingsinclude methods of enhancing NAD biosynthesis in a subject throughadministration of NMN. In some embodiments, the present teachingsinclude methods of promoting NSPC proliferation in a subject, in whichthe methods comprise administration of NMN to the subject. The methodsof each of these embodiments can comprise, consist essentially of, orconsist of administration of a therapeutically effective amount of NMN.

In some embodiments, the present teachings include methods of increasingbone density levels in a subject. In some embodiments, the presentteachings include methods of treating aberrantly low bone density levelsin a subject. In some embodiments, the present teachings include methodsof treating an age-associated bone density decrease in a subject. Insome embodiments, the present teachings include methods of treatingosteoporosis in a subject. In some embodiments, the present teachingsinclude methods of preventing an age-associated bone density decrease ina subject. The methods of each of these embodiments can comprise,consist essentially of, or consist of administration of atherapeutically effective amount of NMN.

In various embodiments, the inventors disclose that photoreceptorneuronal cell death and vision can be rescued by NMN administration. Invarious embodiments, the present inventors demonstrate that nicotinamidephosphoribosyl transferase (NAMPT)-mediated NAD biosynthesis can play arole in for rod and/or cone PR neuron survival. In various embodiments,the present inventors demonstrate that decreased NAD levels can causeimpaired mitochondrial function in PR neurons, alterations in TCA cyclemetabolites, and can lead to cell death and blindness.

In some embodiments, the inventors have demonstrated that deleting NAMPTcan lead to photoreceptor death, loss of normal retinal structure andfunction, and vision loss. In some embodiments, the inventors havedemonstrated that such damage to photoreceptor neurons and theirfunction can be reversed with supplementation of nicotinamidemononucleotide (NMN), an NAMPT enzymatic reaction product. In someembodiments, the present teachings include NMN administration to restoreNAD levels in the retina. In some embodiments, NMN supplementation canbe an effective therapeutic intervention for many retinal degenerativediseases. Without being limited by theory, NMN supplementation canrestore retinal NAD levels.

In some embodiments, the present inventors have demonstrated in vivousing mouse models and in vitro using cell lines that photoreceptordeath can be prevented by NMN supplementation. In some embodiments,methods of NMN supplementation for the prevention/treatment of manyretinal degenerative diseases are disclosed.

Accordingly, in various embodiments, the inventors disclose methods ofpreventing, methods of reducing risk of, and methods of treating variousdiseases associated with photoreceptor dysfunction, including, withoutlimitation, age-related macular degeneration (AMD), inherited andacquired retinal diseases such as, without limitation, retinitispigmentosa (RP), rod and cone dystrophism, and Leber's congenitalamaurosis (LCA) by administration of NMN. In various embodiments, NMNadministration can be an effective intervention for the preventionand/or treatment of orphan retinal degenerative diseases including butnot limited to rod dystrophy, cone dystrophy, retinitis pigmentosa,other inherited retinal degenerations, Leber's congenital amaurosis(LCA) and acquired retinal degenerations such as, but not limited to,age-related macular degeneration photoreceptor degeneration followingretinal detachment.

In various embodiments, NMN can be administered by any administrationroute known to skilled artisans, such as, without limitation, oral,parenteral, intraocular, intraperitoneal, intravenous or intramuscularroutes. In various embodiments, NMN can be administered with or withoutan excipient.

In some embodiments, the present teachings include methods of treatingmacular degeneration in a subject. In some embodiments, the presentteachings include methods of treating aberrant retinal NAD levels in asubject, including aberrantly low retinal NAD levels. In someembodiments, the present teachings include methods of treating retinaldegeneration in a subject. In some embodiments, the present teachingsinclude methods of treating photoreceptor damage in a subject. In someembodiments, the present teachings include methods of treatingphotoreceptor degeneration in a subject. In some embodiments, thepresent teachings include methods of treating vision loss associatedwith retinal degeneration in a subject. In some embodiments, the presentteachings include methods of treating vision loss in a subject. In someembodiments, the present teachings include methods of treating aberrantretinal structure in a subject. In some embodiments, the presentteachings include methods of treating aberrant retinal function in asubject. In some embodiments, the present teachings include methods oftreating aberrant retinal function in a subject. In some embodiments,the present teachings include methods of treating aberrant retinalfunction in a subject. In some embodiments, the present teachingsinclude methods of increasing retinal NAD levels in a subject. In someembodiments, the present teachings include methods of reducing risk ofdeveloping macular degeneration in a subject. In some embodiments, thepresent teachings include methods of reducing risk of developing maculardegeneration in a subject. In some embodiments, the present teachingsinclude methods of reducing risk of developing aberrant retinal NADlevels in a subject. In some embodiments, the present teachings includemethods of reducing risk of developing retinal degeneration in asubject. In some embodiments, the present teachings include methods ofreducing risk of developing photoreceptor damage/degeneration in asubject. In some embodiments, the present teachings include methods ofreducing risk of developing vision loss associated with retinaldegeneration in a subject. In some embodiments, the present teachingsinclude methods of reducing risk of developing vision loss in a subject.In some embodiments, the present teachings include methods of reducingrisk of developing aberrant retinal structure in a subject. In someembodiments, the present teachings include methods of reducing risk ofdeveloping aberrant retinal structure in a subject. In some embodiments,the present teachings include methods of reducing risk of developingaberrant retinal function in a subject. In some embodiments, the presentteachings include methods of reducing risk of developing aberrantretinal function in a subject. In some embodiments, the presentteachings include methods of treating a photoreceptor dysfunction in asubject. In some embodiments, the present teachings include methods oftreating a retina disease in a subject. In various embodiments, thesemethods can comprise, consist essentially of, or consist ofadministering to a subject a pharmaceutically effective amount ofnicotinamide mononucleotide (NMN). In some embodiments, apharmaceutically effective amount of nicotinamide mononucleotide (NMN)can be an amount effective for increasing retinal NAD levels. In someembodiments a retina disease that can be treated by administration ofNMN can be retinitis pigmentosa (RP), Leber's congenital amaurosis(LCA), rod dystrophy, cone dystrophy, rod-cone dystrophy, cone-roddystrophy, age-related macular degeneration, photoreceptor degenerationfollowing retinal detachments, or a combination thereof.

In various embodiments, these methods can each independently comprise,consist essentially of, or consist of administering to a subject apharmaceutically effective amount of nicotinamide mononucleotide (NMN).In some embodiments, NMN can be administered at a dosage rate of about100 mg per day, from 100 mg per day to 2000 mg per day, or about 2000 mgper day. In some embodiments, NMN can be administered at a dosage rateof 0.5 mg, about 0.5 mg, 1 mg, about 1 mg, 5 mg, about 5 mg, 10 mg,about 10 mg, 20 mg, about 20 mg, 30 mg, about 30 mg, 40 mg, about 40 mg,50 mg, about 50 mg, 60 mg, about 60 mg, 70 mg, about 70 mg, 80 mg, about80 mg, 90 mg, about 90 mg, about 100 mg per day, 100 mg per day, 150 mg,about 150 mg, about 200 mg per day, 200 mg per day, about 300 mg perday, 300 mg per day, about 400 mg per day, 400 mg per day, about 500 mgper day, 500 mg per day, about 600 mg per day, 600 mg per day, about 700mg per day, 700 mg per day, about 800 mg per day, 800 mg per day, about900 mg per day, 900 mg per day, about 1000 mg per day, 1000 mg per day,about 1100 mg per day, 1100 mg per day, about 1200 mg per day, 1200 mgper day, about 1300 mg per day, 1300 mg per day, 1350 mg, about 1350 mg,about 1400 mg per day, 1400 mg per day, about 1500 mg per day, 1500 mgper day, about 1600 mg per day, 1600 mg per day, about 1700 mg per day,1700 mg per day, about 1800 mg per day, 1800 mg per day, about 1900 mgper day, 1900 mg per day, about 2000 mg per day, 2000 mg per day, 2040mg, about 2040 mg, 2250 mg, about 2250 mg, 2260 mg, about 2260 mg, 2700mg, about 2700 mg, 2720 mg, about 2720 mg, 3400 mg, about 3400 mg, 3390mg, about 3390 mg, 3400 mg, about 3400 mg, 3600 mg, about 3600 mg, 4080mg, about 4080 mg, 4500 mg, about 4500 mg, 4520 mg, about 4520 mg, 5440mg, about 5440 mg, 5650 mg, about 5650 mg, 6800 mg, about 6800 mg, or analternation or combination thereof. In some embodiments, NMN can beadministered at a dosage rate of about 100 mg/kg body weight/day, from100 mg/kg body weight/day to 500 mg/kg body weight/day, or about 500mg/kg body weight/day. In some embodiments, NMN can be administered at adosage rate of about 100 mg/kg body weight/day, from 100 mg/kg bodyweight/day to 300 mg/kg body weight/day, or about 300 mg/kg bodyweight/day. In some embodiments, these methods can compriseadministering to a subject any of the pharmaceutically acceptablecompositions of the present teachings. In some embodiments, thesemethods can comprise, consist essentially of or consist of administeringa formulation once per day. In some embodiments, these methods cancomprise, consist essentially of or consist of administering aformulation twice per day, three times per day, or four times per day.In some embodiments, these methods can comprise, consist essentially ofor consist of administering a sustained-release formulation once, or atlong intervals such as, without limitation, once per week, semi-weekly,or once per month.

In various embodiments, these methods can each independently comprise,consist essentially of, or consist of administering to a subject apharmaceutically effective amount of a formulation comprisingnicotinamide mononucleotide (NMN) such as, without limitation, a pill, atablet, a caplet, a capsule, a chewable tablet, a quick dissolve tablet,a powder, an effervescent tablet, a hard gelatin capsule, a soft gelatincapsule, a non-aqueous liquid, an aqueous liquid, a granule, a capsule,a suspension, a solution, an emulsion, a syrup, a sterilized aqueoussuspension or solution, a non-aqueous suspension or solution, alyophilized formulation, a suppository or a food product. In variousembodiments, these methods can each independently comprise, consistessentially of, or consist of administering to a subject apharmaceutically effective amount of nicotinamide mononucleotide by oraladministration, sublingual administration, parenteral administration,administration by injection, subcutaneous injection, intramuscularinjection, intraperitoneal injection, intra-ocular injection, directocular contact (eye drops) or a combination thereof.

In various embodiments, a subject can be a mammal. In variousembodiments, a subject can be a vertebrate, such as a mammal, a fish, abird or a reptile. A mammal can be, without limitation, a human, arodent, a canine, a feline, a bovine, an ovine, an equine or a porcine.In some embodiments, a subject can be a bird such as a chicken, areptile, a fish, or other aquatic organism.

In some embodiments, the present teachings include methods ofaugmentation of NAD+ levels during aging with NMN administration tomaintain an NSPC pool, through administration of NMN. In someembodiments, the present teachings include methods of enhancing NAD+levels in NSPCs in a subject through administration of NMN, to preservean endogenous NSPC population for the repair of aged, diseased, ordamaged brain.

The present teachings include the following non-limiting aspects.

1. A pharmaceutically acceptable composition comprising, consistingessentially of, or consisting of: nicotinamide mononucleotide (NMN), asalt thereof and/or a prodrug thereof; and

at least one pharmaceutically acceptable excipient.

2. A pharmaceutically acceptable composition in accordance with aspect1, comprising, consisting essentially of, or consisting of a singledosage formulation.

3. A pharmaceutically acceptable composition in accordance with aspect2, wherein the single dosage formulation is selected from the groupconsisting of a pill, a tablet, a caplet, a capsule, a chewable tablet,a quick dissolve tablet, a powder, an effervescent tablet, a hardgelatin capsule, a soft gelatin capsule, a non-aqueous liquid, anaqueous liquid and an emulsion.4. A pharmaceutically acceptable composition in accordance with aspect2, wherein the single dosage formulation comprises an enteric coating.5. A pharmaceutically acceptable composition in accordance with aspect2, wherein the single dosage formulation comprises, consists essentiallyof, or consists of NMN, a salt thereof and/or a prodrug thereof in anamount of about 100 mg, from 100 mg to 2000 mg, or about 2000 mg.6. A pharmaceutically acceptable composition in accordance with aspect2, wherein the single dosage formulation comprises, consists essentiallyof, or consists of NMN, a salt thereof and/or a prodrug thereof in anamount selected from the group consisting of about 100 mg, about 200 mg,about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg,about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg,about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700mg about 1800 mg, about 1900 mg and about 2000 mg.7. A pharmaceutically acceptable composition in accordance with aspect2, wherein the single dosage formulation comprises, consists essentiallyof, or consists of NMN, a salt thereof and/or the prodrug thereof in anamount selected from the group consisting of 50-150 mg, 151-250 mg,251-350 mg, 351-450 mg, 451-550 mg, 561-650 mg, 651-750 mg, 751-860 mg,861-950 mg, 951-1050 mg, 1051-1150 mg, 1151-1250 mg, 1251-1350 mg,1351-1450 mg, 1451-1550 mg, 1551-1650 mg, 1651-1750 mg, 1751-1850 mg,1851-1950 mg and 1951-2000 mg.8. A pharmaceutically acceptable composition in accordance with aspect2, wherein the single dosage formulation comprises nicotinamidemononucleotide (NMN), a salt thereof and/or a prodrug thereof in anamount selected from the group consisting of 0.5 mg, about 0.5 mg, 1 mg,about 1 mg, 5 mg, about 5 mg, 10 mg, about 10 mg, 20 mg, about 20 mg, 30mg, about 30 mg, 40 mg, about 40 mg, 50 mg, about 50 mg, 60 mg, about 60mg, 70 mg, about 70 mg, 80 mg, about 80 mg, 90 mg, about 90 mg, 100 mg,about 100 mg, 150 mg, about 150 mg, 200 mg, about 200 mg, 300 mg, about300 mg, 400 mg, about 400 mg, 450 mg, about 450 mg, 500 mg, about 500mg, 600 mg, about 600 mg, 680 mg, about 680 mg, 700 mg, about 700 mg,800 mg, about 800 mg, 900 mg, about 900 mg, 1000 mg, about 1000 mg, 1100mg, about 1100 mg, 1130 mg, about 1130 mg, 1200 mg, about 1200 mg, 1300mg, about 1300 mg, 1350 mg, about 1350 mg, 1360 mg, about 1360 mg, 1400mg, about 1400 mg, 1500 mg, about 1500 mg, 1600 mg, about 1600 mg, 1700mg, about 1700 mg, 1800 mg, about 1800 mg, 2040 mg, about 2040 mg, 2250mg, about 2250 mg, 2260 mg, about 2260 mg, 2700 mg, about 2700 mg, 2720mg, about 2720 mg, 3400 mg, about 3400 mg, 3390 mg, about 3390 mg, 3400mg, about 3400 mg, 3600 mg, about 3600 mg, 4080 mg, about 4080 mg, 4500mg, about 4500 mg, 4520 mg, about 4520 mg, 5440 mg, about 5440 mg, 5650mg, about 5650 mg, 6800 mg, and about 6800 mg.9. A pharmaceutically acceptable composition in accordance with aspect2, wherein the single dosage formulation comprises a liquid solution, anemulsion or a suspension.10. A pharmaceutically acceptable composition in accordance with aspect2, wherein the single dosage formulation is a liquid solution, anemulsion or a suspension.11. A pharmaceutically acceptable composition in accordance with aspect2, wherein the single dosage formulation comprises a food product.12. A pharmaceutically acceptable composition in accordance with aspect2, wherein the single dosage formulation is a food product.13. A pharmaceutically acceptable composition in accordance with aspect1, wherein the composition is suitable for oral administration.14. A pharmaceutically acceptable composition in accordance with aspect1, wherein the composition is suitable for sublingual administration.15. A pharmaceutically acceptable composition in accordance with aspect1, wherein the composition is suitable for parenteral administration.16. A pharmaceutically acceptable composition in accordance with aspect1, wherein the composition is suitable for administration by injection.17. A pharmaceutically acceptable composition in accordance with aspect1, wherein the composition is suitable for administration bysubcutaneous injection.18. A pharmaceutically acceptable composition in accordance with aspect1, wherein the composition is suitable for administration byintramuscular injection.19. A pharmaceutically acceptable composition in accordance with aspect1, wherein the composition is suitable for administration byintraperitoneal injection.20. A pharmaceutically acceptable composition in accordance with aspect1, wherein the composition is suitable for administration byintra-ocular injection or topically to the eye (eye drops).21. A pharmaceutically acceptable composition in accordance with aspect1, wherein the composition is selected from the group consisting of atablet, a pill, powder, granules, a capsule, a suspension, a solution,an emulsion, a syrup, a sterilized aqueous suspension or solution, anon-aqueous suspension or solution, a lyophilized formulation, and asuppository.22. A pharmaceutically acceptable composition in accordance with aspect1, wherein the at least one excipient is selected from the groupconsisting of a bulking agent, a tableting agent, a dissolution agent, awetting agent, a lubricant, a coloring, a flavoring, a disintegrant, acoating, a binder, an antioxidant, a taste masking agent and asweetener.23. A pharmaceutically acceptable composition in accordance with aspect22, wherein the bulking agent is selected from the group consisting ofmannitol, sorbitol, sucrose and trehalose.24. A pharmaceutically acceptable composition in accordance with aspect1, wherein the composition is formulated as an orally disintegratingcapsule, tablet, pill or wafer.25. A pharmaceutically acceptable composition in accordance with aspect1, wherein the composition is formulated as a liquid, syrup, or spray.26. A pharmaceutically acceptable composition in accordance with aspect1, further comprising at least one vitamin or nutrient selected from thegroup consisting of vitamin C, vitamin D3, vitamin E, vitamin B1,vitamin B2, niacin, vitamin B6, folic acid, vitamin B12, pantothenicacid, biotin, magnesium, zinc, copper, selenium, chromium, alpha lipoicacid, b co-enzyme Q-10, lutein and lycopene.27. A pharmaceutically acceptable composition in accordance with aspect1, further comprising at least one vitamin or nutrient selected from thegroup consisting of from about 150 mg to about 750 mg of vitamin C, fromabout 315 IUs to about 1800 IUs of vitamin D3, from about 75 IUs toabout 150 IUs of vitamin E, from about 15 mg to about 35 mg of vitaminB1, from about 1.7 mg to about 5.1 mg of vitamin B2, from about 20 mg toabout 50 mg of niacin, from about 20 mg to about 50 mg of vitamin B6,from about 0.5 mg to about 2.5 mg of folic acid, from about 35 mcg toabout 105 mcg of vitamin B12, from about 2.5 mg to about 7.5 mg ofpantothenic acid, from about 50 mcg to about 450 mcg of biotin, fromabout 15 mg to about 55 mg of magnesium, from about 15 mg to about 55 mgof zinc, from about 0.5 to about 1.5 mg of copper, from about 75 mcg toabout 175 mcg of selenium, from about 75 mcg to about 225 mcg ofchromium, from about 10 mg to about 40 mg of alpha lipoic acid, fromabout 20 mg to about 50 mg of co-enzyme Q-10, from about 350 mcg toabout 3 mg of lutein and from about 100 mcg to about 750 mcg oflycopene.28. A pharmaceutically acceptable composition in accordance with aspect1, further comprising at least one vitamin or nutrient selected from thegroup consisting of about 500 mg of ascorbic acid; about 400 IUs ofcholecalciferol; about 125 IUs of d-alpha tocopherol succinate; about 35mg of thiamine mononitrate; about 5.1 mg of riboflavin; about 50 mg ofniacinamide; about 50 mg of pyridoxine HCl; about 2.5 mg of folic acid;about 105 mcg of cyanocobalamin; about 7.5 mg of d-calcium pantothenate;about 75 mcg of d-biotin; about 55 mg of dimagnesium malate; about 55 mgof zinc bisglycinate chelate; about 1.5 mg of copper amino acid chelate;about 175 mcg of selenium amino acid chelate; about 225 mcg of chromiumamino acid chelate; about 10 mg of alpha lipoic acid; about 50 mg ofco-enzyme Q-10; about 400 mcg of lutein; about 125 mcg of lycopene.29. A pharmaceutically acceptable composition in accordance with aspect1, wherein the at least one excipient is selected from the groupconsisting of dicalcium phosphate, microcrystalline cellulose, stearicacid, croscarmellose sodium, magnesium trisilicate, magnesium stearate,hydroxypropyl methylcellulose, hypromellose, titanium dioxide,tripotassium citrate, polyvinyl alcohol, fumed silica, citric acid,polyethylene glycol, talc, and any combination thereof.30. A pharmaceutically acceptable composition in accordance with aspect1, wherein the at least one excipient is selected from the groupconsisting of from about 100 mg to about 300 mg of dicalcium phosphate;from about 25 mg to about 75 mg of microcrystalline cellulose; fromabout 10 mg to about 30 mg of stearic acid; from about 10 mg to about 30mg of croscarmellose sodium; from about 5 mg to about 15 mg of magnesiumtrisilicate; from about 5 mg to about 15 mg of magnesium stearate; andfrom about 5 mg to about 15 mg of hydroxypropyl methylcellulose.31. A pharmaceutically acceptable composition in accordance with aspect1, wherein the at least one excipient is selected from the groupconsisting of about 200 mg of dicalcium phosphate; about 50 mg ofmicrocrystalline cellulose; about 20 mg of stearic acid; about 20 mg ofcroscarmellose sodium; about 10 mg of magnesium trisilicate; about 10 mgof magnesium stearate; and about 10 mg of hydroxypropyl methylcellulose.32. A sustained release formulation of nicotinamide mononucleotide fororal administration to a subject, comprising nicotinamide mononucleotideas an active ingredient that is released from the formulation along apre-determined release profile, wherein the formulation comprises anextended release component and an immediate release component, whereinthe extended release component is contained in at least one populationof beads and releases nicotinamide mononucleotide in a continuous mannerand each bead population is coated with its own release controllingcoating and characterized by its own rate of release.33. The formulation of aspect 32, wherein the extended release componentreleases the nicotinamide mononucleotide in vivo in a continuous manner,and 80% of the nicotinamide mononucleotide is released in vitro in aperiod of time selected from not more than 24 hours, not more than 16hours, not more than 12 hours, not more than 8 hours and not more than 4hours.34. The formulation of aspect 32, wherein the immediate releasecomponent is an enhanced immediate release (EIR) composition comprisinga complexing agent, an enhancing agent, or both.35. The formulation of aspect 34, wherein the EIR composition exhibitsan in vitro release profile such that 80% of the active ingredient isdissolved in not more than 30 min.36. The formulation of aspect 35, wherein the EIR composition exhibitsan in vitro release profile selected from a group consisting of: a) adissolution of at least 50% of the active compound in not more than 10minutes; b) a dissolution of at least 70% of the active compound in notmore than 10 minutes; c) a dissolution of at least 25% of the activecompound in not more than 5 minutes; d) a dissolution of at least 40% ofthe active compound in not more than 5 minutes; and e) a dissolution ofat least 55% of the active compound in not more than 5 minutes.37. The formulation of aspect 34, wherein the complexing agent is acyclodextrin selected from a group consisting ofhydroxypropyl-beta-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin,alpha-cyclodextrin, and derivatives thereof.38. The formulation of aspect 34, wherein the enhancing agent isselected from a group comprising solubility enhancing agents,dissolution enhancing agents, absorption enhancing agents, penetrationenhancing agents, surface active agents, stabilizers, enzyme inhibitors,p-glycoprotein inhibitors, multidrug resistance protein inhibitors andcombinations thereof.39. The formulation of aspect 38, wherein the enhancing agent isselected from a group consisting of Vitamin E TPGS, glutamic acid,glycine, sorbitol, mannose, amylose, maltose, mannitol, lactose,sucrose, glucose, xylitose, dextrins, glycerolpolyethylene glycoloxystearate, PEG-32 glyceryl palmitostearate, sodium lauryl sulfate,polyoxyethylene sorbitan monooleate, benzyl alcohol, sorbitanmonolaurate, Poloxamer 407, PEG3350, PVP K25, oleic acid, glycerylmonooleate, sodium benzoate, cetyl alcohol, sucrose stearate,crospovidone, sodium starch glycolate, croscarmellose sodium,carboxymethylcellulose, starch, pregelatinized starch, HPMC, substitutedhydroxypropylcellulose, microcrystalline cellulose sodium bicarbonate,calcium citrate, sodium docusate, menthol and any combination thereof.40. The formulation of aspect 32, wherein at least a part of the activeingredient is in a form of micronized particles.41. The formulation of aspect 40, wherein the particles have an averagesize of from about 2 μm to about 100 μm.42. The formulation of aspect 32, wherein a specific amount of eachcomponent is determined according to the purpose of administration andthe pre-determined release profile, and the total amount of NMN in theformulation is from 0.5 to 3000 mg.43. The formulation of aspect 32, wherein the beads comprise an inertcarrier, NMN, an optional enhancer, and a release controlling coatingthat comprises a coating material and optionally a pore former and otherexcipients.44. The formulation of aspect 43, wherein the inert carrier is selectedfrom a group consisting of cellulose spheres, silicon dioxide, starchand sugar spheres.45. The formulation of aspect 43, wherein the enhancer is selected froma group consisting of solubility enhancers, dissolution enhancers,permeability enhancers, stabilizers, complexing agents, enzymeinhibitors, p-glycoprotein inhibitors, multidrug resistance proteininhibitors and combinations thereof.46. The formulation of aspect 43, wherein the coating material isselected from a group consisting of ethylcellulose, methylcellulose,hydroxypropyl cellulose, hydroxypropylmethyl cellulose, celluloseacetate, cellulose acetate phthalate, polyvinyl alcohol, polyacrylates,polymethacrylates and copolymers thereof; and/or the pore former isselected from a group consisting of glucose, fructose, mannitol,mannose, galactose, sorbitol, pullulan, dextran, water-solublehydrophilic polymers, hydroxyalkylcelluloses, carboxyalkylcelluloses,hydroxypropylmethylcellulose, cellulose ethers, acrylic resins,polyvinylpyrrolidone, cross-linked polyvinylpyrrolidone, polyethyleneoxide, CARBOWAXES™ (Dow Chemical), CARBOPOL) (The Lubrizol Corp.),diols, polyols, polyhydric alcohols, polyalkylene glycols, polyethyleneglycols, polypropylene glycols or block polymers thereof, polyglycols,poly(a-w)alkylenediols; inorganic compounds selected from a groupconsisting of alkali metal salts and alkaline earth metal salts, andcombinations thereof.47. The formulation of aspect 32, wherein an amount of each beadpopulation is determined according to a pre-determined release profile.48. The formulation of aspect 32, wherein the pre-determined releaseprofile comprises a sustained rate of release after an initial immediaterelease.49. The formulation of aspect 32, suitable for once a day oraladministration.50. The formulation of aspect 32, wherein at least one population ofbeads consists of extended release NMN beads additionally comprising animmediate release component coated on top of the release controllingcoating.51. The formulation of aspect 43, wherein the enhancer is contained in alayer separate from the release controlling coating.52. The formulation of aspect 32, additionally comprising at least oneenhancing agent, wherein the enhancing agent is incorporated into theformulation in the form of a powder or of a population of beads that areoptionally characterized by a controlled rate of release, and whereinthe enhancing agent is separated from the active ingredient.53. A method of treating, ameliorating, mitigating, slowing, arresting,preventing or reversing age-associated obesity in a subject, comprising:administering to a subject a pharmaceutically effective amount ofnicotinamide mononucleotide (NMN).54. A method in accordance with aspect 53, wherein the NMN isadministered at a dosage rate of about 100 mg per day, from 100 mg perday to 2000 mg per day, or about 2000 mg per day.55. A method in accordance with aspect 53, wherein the NMN isadministered at a dosage rate of about 100 mg per day, or 100 mg perday.56. A method in accordance with aspect 53, wherein the NMN isadministered at a dosage rate of about 200 mg per day, or 200 mg perday.57. A method in accordance with aspect 53, wherein the NMN isadministered at a dosage rate of about 300 mg per day, or 300 mg perday.58. A method in accordance with aspect 53, wherein the NMN isadministered at a dosage rate of about 400 mg per day, or 400 mg perday.59. A method in accordance with aspect 53, wherein the NMN isadministered at a dosage rate of about 500 mg per day, or 500 mg perday.60. A method in accordance with aspect 53, wherein the NMN isadministered at a dosage rate of about 600 mg per day, or 600 mg perday.61. A method in accordance with aspect 53, wherein the NMN isadministered at a dosage rate of about 700 mg per day, or 700 mg perday.62. A method in accordance with aspect 53, wherein the NMN isadministered at a dosage rate of about 800 mg per day, or 800 mg perday.63. A method in accordance with aspect 53, wherein the NMN isadministered at a dosage rate of about 900 mg per day, or 900 mg perday.64. A method in accordance with aspect 53, wherein the NMN isadministered at a dosage rate of about 1,000 mg per day, or 1,000 mg perday.65. A method in accordance with aspect 53, wherein the NMN isadministered at a dosage rate of about 1,100 mg per day, or 1,100 mg perday.66. A method in accordance with aspect 53, wherein the NMN isadministered at a dosage rate of about 1,200 mg per day, or 1,200 mg perday.67. A method in accordance with aspect 53, wherein the NMN isadministered at a dosage rate of about 1,300 mg per day, or 1,300 mg perday.68. A method in accordance with aspect 53, wherein the NMN isadministered at a dosage rate of about 1,400 mg per day, or 1,400 mg perday.69. A method in accordance with aspect 53, wherein the NMN isadministered at a dosage rate of about 1,500 mg per day, or 1,500 mg perday.70. A method in accordance with aspect 53, wherein the NMN isadministered at a dosage rate of about 1,600 mg per day, or 1,600 mg perday.71. A method in accordance with aspect 53, wherein the NMN isadministered at a dosage rate of about 1,700 mg per day, or 1,700 mg perday.72. A method in accordance with aspect 53, wherein the NMN isadministered at a dosage rate of about 1,800 mg per day, or 1,800 mg perday.73. A method in accordance with aspect 53, wherein the NMN isadministered at a dosage rate of about 1,900 mg per day, or 1,990 mg perday.74. A method in accordance with aspect 53, wherein the NMN isadministered at a dosage rate of about 2,000 mg per day, or 2,000 mg perday.75. A method in accordance with aspect 53, wherein the NMN isadministered at a dosage rate of about 100 mg/kg body weight/day, from100 mg/kg body weight/day to 300 mg/kg body weight/day, or about 300mg/kg body weight/day.76. A method in accordance with aspect 53, comprising administering to asubject a formulation of any one of aspects 1-52.77. A method in accordance with aspect 76, wherein the administering aformulation comprises, consists essentially of or consists ofadministering the formulation once per day.78. A method in accordance with aspect 76, wherein the administering aformulation comprises, consists essentially of or consists ofadministering the formulation twice per day.79. A method of treating, ameliorating, mitigating, slowing, arresting,preventing or reversing age-associated increases in blood lipid levelsin a subject, comprising: administering to a subject a pharmaceuticallyeffective amount of nicotinamide mononucleotide (NMN).80. A method in accordance with aspect 79, wherein the NMN isadministered at a dosage rate of about 100 mg per day, from 100 mg perday to 2000 mg per day, or about 2000 mg per day.81. A method in accordance with aspect 79, wherein the NMN isadministered at a dosage rate of about 100 mg per day, or 100 mg perday.82. A method in accordance with aspect 79, wherein the NMN isadministered at a dosage rate of about 200 mg per day, or 200 mg perday.83. A method in accordance with aspect 79, wherein the NMN isadministered at a dosage rate of about 300 mg per day, or 300 mg perday.84. A method in accordance with aspect 79, wherein the NMN isadministered at a dosage rate of about 400 mg per day, or 400 mg perday.85. A method in accordance with aspect 79, wherein the NMN isadministered at a dosage rate of about 500 mg per day, or 500 mg perday.86. A method in accordance with aspect 79, wherein the NMN isadministered at a dosage rate of about 600 mg per day, or 600 mg perday.87. A method in accordance with aspect 79, wherein the NMN isadministered at a dosage rate of about 700 mg per day, or 700 mg perday.88. A method in accordance with aspect 79, wherein the NMN isadministered at a dosage rate of about 800 mg per day, or 800 mg perday.89. A method in accordance with aspect 79, wherein the NMN isadministered at a dosage rate of about 900 mg per day, or 900 mg perday.90. A method in accordance with aspect 79, wherein the NMN isadministered at a dosage rate of about 1,000 mg per day, or 1,000 mg perday.91. A method in accordance with aspect 79, wherein the NMN isadministered at a dosage rate of about 1,100 mg per day, or 1,100 mg perday.92. A method in accordance with aspect 79, wherein the NMN isadministered at a dosage rate of about 1,200 mg per day, or 1,200 mg perday.93. A method in accordance with aspect 79, wherein the NMN isadministered at a dosage rate of about 1,300 mg per day, or 1,300 mg perday.94. A method in accordance with aspect 79, wherein the NMN isadministered at a dosage rate of about 1,400 mg per day, or 1,400 mg perday.95. A method in accordance with aspect 79, wherein the NMN isadministered at a dosage rate of about 1,500 mg per day, or 1,500 mg perday.96. A method in accordance with aspect 79, wherein the NMN isadministered at a dosage rate of about 1,600 mg per day, or 1,600 mg perday.97. A method in accordance with aspect 79, wherein the NMN isadministered at a dosage rate of about 1,700 mg per day, or 1,700 mg perday.98. A method in accordance with aspect 79, wherein the NMN isadministered at a dosage rate of about 1,800 mg per day, or 1,800 mg perday.99. A method in accordance with aspect 79, wherein the NMN isadministered at a dosage rate of about 1,900 mg per day, or 1,900 mg perday.100. A method in accordance with aspect 79, wherein the NMN isadministered at a dosage rate of about 2,000 mg per day, or 2,000 mg perday.101. A method in accordance with aspect 79, wherein the NMN isadministered at a dosage rate of about 100 mg/kg body weight/day, from100 mg/kg body weight/day to 300 mg/kg body weight/day, or about 300mg/kg body weight/day.102. A method in accordance with aspect 79, comprising administering toa subject a formulation of any one of aspects 1-51.103. A method in accordance with aspect 102, wherein the administering aformulation comprises, consists essentially of or consists ofadministering the formulation once per day.104. A method in accordance with aspect 102, wherein the administering aformulation comprises, consists essentially of or consists ofadministering the formulation twice per day.105. A method of treating, ameliorating, mitigating, slowing, arresting,preventing or reversing age-associated loss of insulin sensitivity in asubject, comprising: administering to a subject a pharmaceuticallyeffective amount of nicotinamide mononucleotide (NMN).106. A method in accordance with aspect 105, wherein the NMN isadministered at a dosage rate of about 100 mg per day, from 100 mg perday to 2000 mg per day, or about 2000 mg per day.107. A method in accordance with aspect 105, wherein the NMN isadministered at a dosage rate of about 100 mg per day, or 100 mg perday.108. A method in accordance with aspect 105, wherein the NMN isadministered at a dosage rate of about 200 mg per day, or 200 mg perday.109. A method in accordance with aspect 105, wherein the NMN isadministered at a dosage rate of about 300 mg per day, or 300 mg perday.110. A method in accordance with aspect 105, wherein the NMN isadministered at a dosage rate of about 400 mg per day, or 400 mg perday.111. A method in accordance with aspect 105, wherein the NMN isadministered at a dosage rate of about 500 mg per day, or 500 mg perday.112. A method in accordance with aspect 105, wherein the NMN isadministered at a dosage rate of about 600 mg per day, or 600 mg perday.113. A method in accordance with aspect 105, wherein the NMN isadministered at a dosage rate of about 700 mg per day, or 700 mg perday.114. A method in accordance with aspect 105, wherein the NMN isadministered at a dosage rate of about 800 mg per day, or 800 mg perday.115. A method in accordance with aspect 105, wherein the NMN isadministered at a dosage rate of about 900 mg per day, or 900 mg perday.116. A method in accordance with aspect 105, wherein the NMN isadministered at a dosage rate of about 1,000 mg per day, or 1,000 mg perday.117. A method in accordance with aspect 105, wherein the NMN isadministered at a dosage rate of about 1,100 mg per day, or 1,100 mg perday.118. A method in accordance with aspect 105, wherein the NMN isadministered at a dosage rate of about 1,200 mg per day, or 1,200 mg perday.119. A method in accordance with aspect 105, wherein the NMN isadministered at a dosage rate of about 1,300 mg per day, or 1,300 mg perday.120. A method in accordance with aspect 105, wherein the NMN isadministered at a dosage rate of about 1,400 mg per day, or 1,400 mg perday.121. A method in accordance with aspect 105, wherein the NMN isadministered at a dosage rate of about 1,500 mg per day, or 1,500 mg perday.122. A method in accordance with aspect 105, wherein the NMN isadministered at a dosage rate of about 1,600 mg per day, or 1,600 mg perday.123. A method in accordance with aspect 105, wherein the NMN isadministered at a dosage rate of about 1,700 mg per day, or 1,700 mg perday.124. A method in accordance with aspect 105, wherein the NMN isadministered at a dosage rate of about 1,800 mg per day, or 1,800 mg perday.125. A method in accordance with aspect 105, wherein the NMN isadministered at a dosage rate of about 1,900 mg per day, or 1,900 mg perday.125. A method in accordance with aspect 105, wherein the NMN isadministered at a dosage rate of about 2,000 mg per day, or 2,000 mg perday.127. A method in accordance with aspect 105, wherein the NMN isadministered at a dosage rate of about 100 mg/kg body weight/day, from100 mg/kg body weight/day to 300 mg/kg body weight/day, or about 300mg/kg body weight/day.128. A method in accordance with aspect 105, comprising administering toa subject a formulation of any one of aspects 1-51.129. A method in accordance with aspect 128, wherein the administering aformulation comprises, consists essentially of or consists ofadministering the formulation once per day.130. A method in accordance with aspect 128, wherein the administering aformulation comprises, consists essentially of or consists ofadministering the formulation twice per day.131. A method of treating, ameliorating, mitigating, slowing, arresting,preventing or reversing age-associated impairment of memory function ina subject, comprising: administering to a subject a pharmaceuticallyeffective amount of nicotinamide mononucleotide (NMN).131. A method in accordance with aspect 130, wherein the NMN isadministered at a dosage rate of about 100 mg per day, from 100 mg perday to 2000 mg per day, or about 2000 mg per day.133. A method in accordance with aspect 131, wherein the NMN isadministered at a dosage rate of about 100 mg per day, or 100 mg perday.134. A method in accordance with aspect 131, wherein the NMN isadministered at a dosage rate of about 200 mg per day, or 200 mg perday.135. A method in accordance with aspect 131, wherein the NMN isadministered at a dosage rate of about 300 mg per day, or 300 mg perday.136. A method in accordance with aspect 131, wherein the NMN isadministered at a dosage rate of about 400 mg per day, or 400 mg perday.137. A method in accordance with aspect 131, wherein the NMN isadministered at a dosage rate of about 500 mg per day, or 500 mg perday.138. A method in accordance with aspect 131, wherein the NMN isadministered at a dosage rate of about 600 mg per day, or 600 mg perday.139. A method in accordance with aspect 131, wherein the NMN isadministered at a dosage rate of about 700 mg per day, or 700 mg perday.140. A method in accordance with aspect 131, wherein the NMN isadministered at a dosage rate of about 800 mg per day, or 800 mg perday.141. A method in accordance with aspect 131, wherein the NMN isadministered at a dosage rate of about 900 mg per day, or 900 mg perday.142. A method in accordance with aspect 131, wherein the NMN isadministered at a dosage rate of about 1,000 mg per day, or 1,000 mg perday.143. A method in accordance with aspect 131, wherein the NMN isadministered at a dosage rate of about 1,100 mg per day, or 1,100 mg perday.144. A method in accordance with aspect 131, wherein the NMN isadministered at a dosage rate of about 1,200 mg per day, or 1,200 mg perday.145. A method in accordance with aspect 131, wherein the NMN isadministered at a dosage rate of about 1,300 mg per day, or 1,300 mg perday.146. A method in accordance with aspect 131, wherein the NMN isadministered at a dosage rate of about 1,400 mg per day, or 1,400 mg perday.147. A method in accordance with aspect 131, wherein the NMN isadministered at a dosage rate of about 1,500 mg per day, or 1,500 mg perday.148. A method in accordance with aspect 131, wherein the NMN isadministered at a dosage rate of about 1,600 mg per day, or 1,600 mg perday.149. A method in accordance with aspect 131, wherein the NMN isadministered at a dosage rate of about 1,700 mg per day, or 1,700 mg perday.150. A method in accordance with aspect 131, wherein the NMN isadministered at a dosage rate of about 1,800 mg per day, or 1,800 mg perday.151. A method in accordance with aspect 131, wherein the NMN isadministered at a dosage rate of about 1,900 mg per day, or 1,900 mg perday.152. A method in accordance with aspect 131, wherein the NMN isadministered at a dosage rate of about 2,000 mg per day, or 2,000 mg perday.153. A method in accordance with aspect 131, wherein the NMN isadministered at a dosage rate of about 100 mg/kg body weight/day, from100 mg/kg body weight/day to 300 mg/kg body weight/day, or about 300mg/kg body weight/day.154. A method in accordance with aspect 131, comprising administering toa subject a formulation of any one of aspects 1-51.155. A method in accordance with aspect 154, wherein the administering aformulation comprises, consists essentially of or consists ofadministering the formulation once per day.156. A method in accordance with aspect 154, wherein the administering aformulation comprises, consists essentially of or consists ofadministering the formulation twice per day.157. A method of treating, ameliorating, mitigating, slowing, arresting,preventing or reversing age-associated decline in eye function in asubject, comprising: administering to a subject a pharmaceuticallyeffective amount of nicotinamide mononucleotide (NMN).158. A method in accordance with aspect 157, wherein the NMN isadministered at a dosage rate of about 100 mg per day, from 100 mg perday to 2000 mg per day, or about 2000 mg per day.159. A method in accordance with aspect 157, wherein the NMN isadministered at a dosage rate of about 100 mg per day, or 100 mg perday.160. A method in accordance with aspect 157, wherein the NMN isadministered at a dosage rate of about 200 mg per day, or 200 mg perday.161. A method in accordance with aspect 157, wherein the NMN isadministered at a dosage rate of about 300 mg per day, or 300 mg perday.162. A method in accordance with aspect 157, wherein the NMN isadministered at a dosage rate of about 400 mg per day, or 400 mg perday.163. A method in accordance with aspect 157, wherein the NMN isadministered at a dosage rate of about 500 mg per day, or 500 mg perday.164. A method in accordance with aspect 157, wherein the NMN isadministered at a dosage rate of about 600 mg per day, or 600 mg perday.165. A method in accordance with aspect 157, wherein the NMN isadministered at a dosage rate of about 700 mg per day, or 700 mg perday.166. A method in accordance with aspect 157, wherein the NMN isadministered at a dosage rate of about 800 mg per day, or 800 mg perday.167. A method in accordance with aspect 157, wherein the NMN isadministered at a dosage rate of about 900 mg per day, or 900 mg perday.168. A method in accordance with aspect 157, wherein the NMN isadministered at a dosage rate of about 1,000 mg per day, or 1,000 mg perday.169. A method in accordance with aspect 157, wherein the NMN isadministered at a dosage rate of about 1,100 mg per day, or 1,100 mg perday.170. A method in accordance with aspect 157, wherein the NMN isadministered at a dosage rate of about 1,200 mg per day, or 1,200 mg perday.171. A method in accordance with aspect 157, wherein the NMN isadministered at a dosage rate of about 1,300 mg per day, or 1,300 mg perday.172. A method in accordance with aspect 157, wherein the NMN isadministered at a dosage rate of about 1,400 mg per day, or 1,400 mg perday.173. A method in accordance with aspect 157, wherein the NMN isadministered at a dosage rate of about 1,500 mg per day, or 1,500 mg perday.174. A method in accordance with aspect 157, wherein the NMN isadministered at a dosage rate of about 1,600 mg per day, or 1,600 mg perday.175. A method in accordance with aspect 157, wherein the NMN isadministered at a dosage rate of about 1,700 mg per day, or 1,700 mg perday.176. A method in accordance with aspect 157, wherein the NMN isadministered at a dosage rate of about 1,800 mg per day, or 1,800 mg perday.177. A method in accordance with aspect 157, wherein the NMN isadministered at a dosage rate of about 1,900 mg per day, or 1,900 mg perday.178. A method in accordance with aspect 157, wherein the NMN isadministered at a dosage rate of about 2,000 mg per day, or 2,000 mg perday.179. A method in accordance with aspect 157, wherein the NMN isadministered at a dosage rate of about 100 mg/kg body weight/day, from100 mg/kg body weight/day to 300 mg/kg body weight/day, or about 300mg/kg body weight/day.180. A method in accordance with aspect 157, comprising administering toa subject a formulation of any one of aspects 1-51.181. A method in accordance with aspect 180, wherein the administering aformulation comprises, consists essentially of or consists ofadministering the formulation once per day.182. A method in accordance with aspect 180, wherein the administering aformulation comprises, consists essentially of or consists ofadministering the formulation twice per day.183. A method of treating, ameliorating, mitigating, slowing, arresting,preventing or reversing age-associated retinal degeneration in asubject, comprising: administering to a subject a pharmaceuticallyeffective amount of nicotinamide mononucleotide (NMN).184. A method in accordance with aspect 183, wherein the NMN isadministered at a dosage rate of about 100 mg per day, from 100 mg perday to 2000 mg per day, or about 2000 mg per day.185. A method in accordance with aspect 183, wherein the NMN isadministered at a dosage rate of about 100 mg per day, or 100 mg perday.186. A method in accordance with aspect 183, wherein the NMN isadministered at a dosage rate of about 200 mg per day, or 200 mg perday.187. A method in accordance with aspect 183, wherein the NMN isadministered at a dosage rate of about 300 mg per day, or 300 mg perday.188. A method in accordance with aspect 183, wherein the NMN isadministered at a dosage rate of about 400 mg per day, or 400 mg perday.189. A method in accordance with aspect 183, wherein the NMN isadministered at a dosage rate of about 500 mg per day, or 500 mg perday.190. A method in accordance with aspect 183, wherein the NMN isadministered at a dosage rate of about 600 mg per day, or 600 mg perday.191. A method in accordance with aspect 183, wherein the NMN isadministered at a dosage rate of about 700 mg per day, or 700 mg perday.192. A method in accordance with aspect 183, wherein the NMN isadministered at a dosage rate of about 800 mg per day, or 800 mg perday.193. A method in accordance with aspect 183, wherein the NMN isadministered at a dosage rate of about 900 mg per day, or 900 mg perday.194. A method in accordance with aspect 183, wherein the NMN isadministered at a dosage rate of about 1,000 mg per day, or 1,000 mg perday.195. A method in accordance with aspect 183, wherein the NMN isadministered at a dosage rate of about 1,100 mg per day, or 1,100 mg perday.196. A method in accordance with aspect 183, wherein the NMN isadministered at a dosage rate of about 1,200 mg per day, or 1,200 mg perday.197. A method in accordance with aspect 183, wherein the NMN isadministered at a dosage rate of about 1,300 mg per day, or 1,300 mg perday.198. A method in accordance with aspect 183, wherein the NMN isadministered at a dosage rate of about 1,400 mg per day, or 1,400 mg perday.199. A method in accordance with aspect 183, wherein the NMN isadministered at a dosage rate of about 1,500 mg per day, or 1,500 mg perday.200. A method in accordance with aspect 183, wherein the NMN isadministered at a dosage rate of about 1,600 mg per day, or 1,600 mg perday.201. A method in accordance with aspect 183, wherein the NMN isadministered at a dosage rate of about 1,700 mg per day, or 1,700 mg perday.202. A method in accordance with aspect 183, wherein the NMN isadministered at a dosage rate of about 1,800 mg per day, or 1,800 mg perday.203. A method in accordance with aspect 183, wherein the NMN isadministered at a dosage rate of about 1,900 mg per day, or 1,900 mg perday.204. A method in accordance with aspect 183, wherein the NMN isadministered at a dosage rate of about 2,000 mg per day, or 2,000 mg perday.205. A method in accordance with aspect 183, wherein the NMN isadministered at a dosage rate of about 100 mg/kg body weight/day, from100 mg/kg body weight/day to 300 mg/kg body weight/day, or about 300mg/kg body weight/day.206. A method in accordance with aspect 183, comprising administering toa subject a formulation of any one of aspects 1-52.207. A method in accordance with aspect 206, wherein the administering aformulation comprises, consists essentially of or consists ofadministering the formulation once per day.208. A method in accordance with aspect 206, wherein the administering aformulation comprises, consists essentially of or consists ofadministering the formulation twice per day.209. A method of treating, ameliorating, mitigating, slowing, arresting,preventing or reversing dry eye, comprising administering to a subject apharmaceutically effective amount of nicotinamide mononucleotide (NMN).210. A method in accordance with aspect 209, wherein the NMN isadministered at a dosage rate of about 100 mg per day, from 100 mg perday to 2000 mg per day, or about 2000 mg per day.211. A method in accordance with aspect 209, wherein the NMN isadministered at a dosage rate of about 100 mg per day, or 100 mg perday.212. A method in accordance with aspect 209, wherein the NMN isadministered at a dosage rate of about 200 mg per day, or 200 mg perday.213. A method in accordance with aspect 209, wherein the NMN isadministered at a dosage rate of about 300 mg per day, or 300 mg perday.214. A method in accordance with aspect 209, wherein the NMN isadministered at a dosage rate of about 400 mg per day, or 400 mg perday.215. A method in accordance with aspect 209, wherein the NMN isadministered at a dosage rate of about 500 mg per day, or 500 mg perday.216. A method in accordance with aspect 209, wherein the NMN isadministered at a dosage rate of about 600 mg per day, or 600 mg perday.217. A method in accordance with aspect 209, wherein the NMN isadministered at a dosage rate of about 700 mg per day, or 700 mg perday.216. A method in accordance with aspect 209, wherein the NMN isadministered at a dosage rate of about 800 mg per day, or 800 mg perday.219. A method in accordance with aspect 209, wherein the NMN isadministered at a dosage rate of about 900 mg per day, or 900 mg perday.220. A method in accordance with aspect 209, wherein the NMN isadministered at a dosage rate of about 1,000 mg per day, or 1,000 mg perday.221. A method in accordance with aspect 209, wherein the NMN isadministered at a dosage rate of about 1,100 mg per day, or 1,100 mg perday.222. A method in accordance with aspect 209, wherein the NMN isadministered at a dosage rate of about 1,200 mg per day, or 1,200 mg perday.223. A method in accordance with aspect 209, wherein the NMN isadministered at a dosage rate of about 1,300 mg per day, or 1,300 mg perday.224. A method in accordance with aspect 209, wherein the NMN isadministered at a dosage rate of about 1,400 mg per day, or 1,400 mg perday.225. A method in accordance with aspect 209, wherein the NMN isadministered at a dosage rate of about 1,500 mg per day, or 1,500 mg perday.226. A method in accordance with aspect 209, wherein the NMN isadministered at a dosage rate of about 1,600 mg per day, or 1,600 mg perday.227. A method in accordance with aspect 209, wherein the NMN isadministered at a dosage rate of about 1,700 mg per day, or 1,700 mg perday.228. A method in accordance with aspect 209, wherein the NMN isadministered at a dosage rate of about 1,800 mg per day, or 1,800 mg perday.229. A method in accordance with aspect 209, wherein the NMN isadministered at a dosage rate of about 1,900 mg per day, or 1,900 mg perday.230. A method in accordance with aspect 209, wherein the NMN isadministered at a dosage rate of about 2,000 mg per day, or 2,000 mg perday.231. A method in accordance with aspect 209, wherein the NMN isadministered at a dosage rate of about 100 mg/kg body weight/day, from100 mg/kg body weight/day to 300 mg/kg body weight/day, or about 300mg/kg body weight/day.232. A method in accordance with aspect 209, comprising administering toa subject a formulation of any one of aspects 1-52.233. A method in accordance with aspect 232, wherein the administering aformulation comprises, consists essentially of or consists ofadministering the formulation once per day.234. A method in accordance with aspect 232, wherein the administering aformulation comprises, consists essentially of or consists ofadministering the formulation twice per day.235. A method of increasing NAD+ levels in a subject, comprising:administering to a subject a pharmaceutically effective amount ofnicotinamide mononucleotide (NMN).236. A method of treating age-associated defects in NSPC functionalityin a subject, comprising: administering to a subject a pharmaceuticallyeffective amount of nicotinamide mononucleotide (NMN).237. A method of maintaining at least one NSPC in a subject, comprising:administering to a subject a pharmaceutically effective amount ofnicotinamide mononucleotide (NMN).238. A method of enhancing NAD biosynthesis in a subject, comprising:administering to a subject a pharmaceutically effective amount ofnicotinamide mononucleotide (NMN).239. A method of promoting NSPC proliferation in a subject, comprising:administering to a subject a pharmaceutically effective amount ofnicotinamide mononucleotide (NMN).240. A method in accordance with any of aspects 235-239, wherein the NMNis administered at a dosage rate of about 100 mg per day, from 100 mgper day to 2000 mg per day, or about 2000 mg per day.241. A method in accordance with any of aspects 235-239, wherein the NMNis administered at a dosage rate of 0.5 mg, about 0.5 mg, 1 mg, about 1mg, 5 mg, about 5 mg, 10 mg, about 10 mg, 20 mg, about 20 mg, 30 mg,about 30 mg, 40 mg, about 40 mg, 50 mg, about 50 mg, 60 mg, about 60 mg,70 mg, about 70 mg, 80 mg, about 80 mg, 90 mg, about 90 mg, 100 mg,about 100 mg, 150 mg, about 150 mg, 200 mg, about 200 mg, 300 mg, about300 mg, 400 mg, about 400 mg, 450 mg, about 450 mg, 500 mg, about 500mg, 600 mg, about 600 mg, 680 mg, about 680 mg, 700 mg, about 700 mg,800 mg, about 800 mg, 910 mg, about 900 mg, 1000 mg, about 1000 mg, 1100mg, about 1100 mg, 1130 mg, about 1130 mg, 1200 mg, about 1200 mg, 1300mg, about 1300 mg, 1350 mg, about 1350 mg, 1360 mg, about 1360 mg, 1400mg, about 1400 mg, 1500 mg, about 1500 mg, 1600 mg, about 1600 mg, 1700mg, about 1700 mg, 1800 mg, about 1800 mg, 2040 mg, about 2040 mg, 2250mg, about 2250 mg, 2260 mg, about 2260 mg, 2700 mg, about 2700 mg, 2720mg, about 2720 mg, 3400 mg, about 3400 mg, 3390 mg, about 3390 mg, 3400mg, about 3400 mg, 3600 mg, about 3600 mg, 4080 mg, about 4080 mg, 4500mg, about 4500 mg, 4520 mg, about 4520 mg, 5440 mg, about 5440 mg, 5650mg, about 5650 mg, 6800 mg, and about 6800 mg.242. A method in accordance with any of aspects 235-239, wherein the NMNis administered at a dosage rate of about 100 mg/kg body weight/day,from 100 mg/kg body weight/day to 300 mg/kg body weight/day, or about300 mg/kg body weight/day.243. A method in accordance with any of aspects 235-239, comprisingadministering to a subject a formulation of any one of aspects 1-52.244. A method in accordance with aspect 243, wherein the administering aformulation comprises, consists essentially of or consists ofadministering the formulation once per day.245. A method in accordance with aspect 243, wherein the administering aformulation comprises, consists essentially of or consists ofadministering the formulation twice per day.246. A method of increasing bone density levels in a subject,comprising: administering to a subject a pharmaceutically effectiveamount of nicotinamide mononucleotide (NMN).247. A method of treating aberrantly low bone density levels in asubject, comprising: administering to a subject a pharmaceuticallyeffective amount of nicotinamide mononucleotide (NMN).248. A method of treating an age-associated bone disorder in a subject,comprising: administering to a subject a pharmaceutically effectiveamount of nicotinamide mononucleotide (NMN).249. A method in accordance with claim 284, wherein the age-associatedbone disorder is osteoporosis.250. A method in accordance with any of aspects 246-249, wherein the NMNis administered at a dosage rate of about 100 mg per day, from 100 mgper day to 2000 mg per day, or about 2000 mg per day.251. A method in accordance with any of aspects 246-249, wherein the NMNis administered at a dosage rate of 0.5 mg, about 0.5 mg, 1 mg, about 1mg, 5 mg, about 5 mg, 10 mg, about 10 mg, 20 mg, about 20 mg, 30 mg,about 30 mg, 40 mg, about 40 mg, 50 mg, about 50 mg, 60 mg, about 60 mg,70 mg, about 70 mg, 80 mg, about 80 mg, 90 mg, about 90 mg, 100 mg,about 100 mg, 150 mg, about 150 mg, 200 mg, about 200 mg, 300 mg, about300 mg, 400 mg, about 400 mg, 450 mg, about 450 mg, 500 mg, about 500mg, 600 mg, about 600 mg, 680 mg, about 680 mg, 700 mg, about 700 mg,800 mg, about 800 mg, 900 mg, about 900 mg, 1000 mg, about 1000 mg, 1100mg, about 1100 mg, 1130 mg, about 1130 mg, 1200 mg, about 1200 mg, 1300mg, about 1300 mg, 1350 mg, about 1350 mg, 1360 mg, about 1360 mg, 1400mg, about 1400 mg, 1500 mg, about 1500 mg, 1600 mg, about 1600 mg, 1700mg, about 1700 mg, 1800 mg, about 1800 mg, 2040 mg, about 2040 mg, 2250mg, about 2250 mg, 2260 mg, about 2260 mg, 2700 mg, about 2700 mg, 2720mg, about 2720 mg, 3400 mg, about 3400 mg, 3390 mg, about 3390 mg, 3400mg, about 3400 mg, 3600 mg, about 3600 mg, 4080 mg, about 4080 mg, 4500mg, about 4500 mg, 4520 mg, about 4520 mg, 5440 mg, about 5440 mg, 5650mg, about 5650 mg, 6800 mg, and about 6800 mg.252. A method in accordance with any of aspects 246-249, wherein the NMNis administered at a dosage rate of about 100 mg/kg body weight/day,from 100 mg/kg body weight/day to 300 mg/kg body weight/day, or about300 mg/kg body weight/day.253. A method in accordance with any of aspects 246-249, comprisingadministering to a subject a formulation of any one of aspects 1-52.254. A method in accordance with aspect 253, wherein the administering aformulation comprises, consists essentially of or consists ofadministering the formulation once per day.255. A method in accordance with aspect 253, wherein the administering aformulation comprises, consists essentially of or consists ofadministering the formulation twice per day.256. A method of treating macular degeneration in a subject, comprisingadministering to a subject a therapeutically effective amount of NMN.257. A method of treating macular degeneration in a subject, comprisingadministering to a subject NMN in an amount effective for increasingretinal NAD levels.258. A method of treating aberrant retinal NAD levels in a subject,comprising administering to a subject NMN in an amount effective forincreasing retinal NAD levels.259. A method of treating retinal degeneration in a subject, comprisingadministering to a subject NMN in an amount effective for increasingretinal NAD levels.260. A method of treating retinal degeneration in a subject, comprisingadministering to a subject a therapeutically effective amount of NMN.261. A method of treating photoreceptor damage/degeneration in asubject, comprising administering to a subject a therapeuticallyeffective amount of NMN.262. A method of treating photoreceptor damage/degeneration in asubject, comprising administering to a subject NMN in an amounteffective for increasing retinal NAD levels.263. A method of treating vision loss associated with retinaldegeneration in a subject, comprising: administering to a subject atherapeutically effective amount of NMN.264. A method of treating vision loss in a subject, comprising:administering to a subject NMN in an amount effective for increasingretinal NAD levels.265. A method of treating aberrant retinal structure in a subject,comprising: administering to a subject a therapeutically effectiveamount of NMN.266. A method of treating aberrant retinal structure in a subject,comprising: administering to a subject NMN in an amount effective forincreasing retinal NAD levels.267. A method of treating aberrant retinal function in a subject,comprising: administering to a subject a therapeutically effectiveamount of NMN.268. A method of treating aberrant retinal function in a subject,comprising: administering to a subject NMN in an amount effective forincreasing retinal NAD levels.269. A method of increasing retinal NAD levels in a subject, comprising:administering to a subject NMN in an amount effective for increasingretinal NAD levels.270. A method of reducing risk of developing macular degeneration in asubject, comprising: administering to a subject NMN in an amounteffective for preventing macular degeneration.271. A method of reducing risk of developing macular degeneration in asubject, comprising: administering to a subject NMN in an amounteffective for increasing retinal NAD levels.272. A method of reducing risk of developing aberrant retinal NAD levelsin a subject, comprising: administering to a subject NMN in an amounteffective for increasing retinal NAD levels.273. A method of reducing risk of developing retinal degeneration in asubject, comprising: administering to a subject NMN in an amounteffective for increasing retinal NAD levels.274. A method of reducing risk of developing photoreceptordamage/degeneration in a subject, comprising: administering to a subjectNMN in an amount effective for preventing photoreceptordamage/degeneration.275. A method of reducing risk of developing photoreceptordamage/degeneration in a subject, comprising: administering to a subjectNMN in an amount effective for increasing retinal NAD levels.276. A method of reducing risk of developing vision loss associated withretinal degeneration in a subject, comprising: administering to asubject NMN in an amount effective for reducing risk of developingvision loss associated with retinal degeneration.277. A method of reducing risk of developing vision loss in a subject,comprising: administering to a subject NMN in an amount effective forincreasing retinal NAD levels.278. A method of reducing risk of developing aberrant retinal structurein a subject, comprising: administering to a subject NMN in an amounteffective for preventing development of aberrant retinal structure.279. A method of reducing risk of developing aberrant retinal structurein a subject, comprising: administering to a subject NMN in an amounteffective for increasing retinal NAD levels.280. A method of reducing risk of developing aberrant retinal functionin a subject, comprising: administering to a subject NMN in an amounteffective for preventing development of aberrant retinal function.281. A method of reducing risk of developing aberrant retinal functionin a subject, comprising: administering to a subject NMN in an amounteffective for increasing retinal NAD levels.282. A method of treating a retina disease in a subject, comprising:administering to a subject a therapeutically effective amount of NMN.283. A method in accordance with aspect 282, wherein the retina diseaseis selected from the group consisting of retinitis pigmentosa (RP),Leber's congenital amaurosis (LCA), rod dystrophy, cone dystrophy,rod-cone dystrophy, cone-rod dystrophy, age-related macular degenerationand photoreceptor degeneration following retinal detachment.284. A method in accordance with any of aspects 256-283, wherein the NMNis administered at a dosage rate of about 100 mg per day, from 100 mgper day to 2000 mg per day, or about 2000 mg per day.285. A method in accordance with any of aspects 256-283, wherein the NMNis administered at a dosage rate of 0.5 mg, about 0.5 mg, 1 mg, about 1mg, 5 mg, about 5 mg, 10 mg, about 10 mg, 20 mg, about 20 mg, 30 mg,about 30 mg, 40 mg, about 40 mg, 50 mg, about 50 mg, 60 mg, about 60 mg,70 mg, about 70 mg, 80 mg, about 80 mg, 90 mg, about 90 mg, 100 mg,about 100 mg, 150 mg, about 150 mg, 200 mg, about 200 mg, 300 mg, about300 mg, 400 mg, about 400 mg, 450 mg, about 450 mg, 500 mg, about 500mg, 600 mg, about 600 mg, 680 mg, about 680 mg, 700 mg, about 700 mg,800 mg, about 800 mg, 900 mg, about 900 mg, 1000 mg, about 1000 mg, 1100mg, about 1100 mg, 1130 mg, about 1130 mg, 1200 mg, about 1200 mg, 1300mg, about 1300 mg, 1350 mg, about 1350 mg, 1360 mg, about 1360 mg, 1400mg, about 1400 mg, 1500 mg, about 1500 mg, 1600 mg, about 1600 mg, 1700mg, about 1700 mg, 1800 mg, about 1800 mg, 2040 mg, about 2040 mg, 2250mg, about 2250 mg, 2260 mg, about 2260 mg, 2700 mg, about 2700 mg, 2720mg, about 2720 mg, 3400 mg, about 3400 mg, 3390 mg, about 3390 mg, 3400mg, about 3400 mg, 3600 mg, about 3600) mg, 4080 mg, about 4080 mg, 4500mg, about 4500 mg, 4520 mg, about 4520 mg, 5440 mg, about 5440 mg, 5650mg, about 5650 mg, 6800 mg, and about 6800 mg.286. A method in accordance with any of aspects 256-283, wherein the NMNis administered at a dosage rate of about 100 mg/kg body weight/day,from 100 mg/kg body weight/day to 300 mg/kg body weight/day, or about300 mg/kg body weight/day.287. A method in accordance with any of aspects 256-283, comprisingadministering to a subject a formulation of any one of aspects 1-52.288. A method in accordance with aspect 283, wherein the administering aformulation comprises, consists essentially of or consists ofadministering the formulation once per day.289. A method in accordance with aspect 283, wherein the administering aformulation comprises, consists essentially of or consists ofadministering the formulation twice per day.290. A method in accordance with any of aspects 32 or 53-289, whereinthe subject is a mammal.291. A method in accordance with any of aspects 32 or 53-289, whereinthe subject is a human.292. A method in accordance with any of aspects 32 or 53-289, whereinthe subject is a vertebrate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates structure of nicotinamide mononucleotide (NMN).

FIG. 2 illustrates age-associated body weight increase.

FIG. 3 illustrates age-associated body weight gain.

FIG. 4 illustrates oxygen consumption in control, 100 and 300 mg/kgNMN-administered mice.

FIG. 5 illustrates energy expenditure in control, 100 and 300 mg/kgNMN-administered mice.

FIG. 6 illustrates respiratory quotient in control, 100 and 300 mg/kgNMN-administered mice.

FIG. 7A-C illustrates blood levels of (A) cholesterol, (B) triglyceridesand (C) free fatty acids shown over 12 months in the control and the 100and 300 mg/kg NMN-administered cohorts.

FIG. 8A-C illustrates body weight-matched blood levels of (A)cholesterol, (B) triglycerides and (C) free fatty acids shown over 12months in the control and the 100 and 300 mg/kg NMN-administeredcohorts.

FIG. 9A-B illustrates insulin tolerance shown in (A) blood glucoselevels and (B) percent glucose changes in control and the 100 mg/kg and300 mg/kg NMN-administered groups at the 12-month time point.

FIG. 10 illustrates freezing responses of regular chow-fed control,HFD-fed, and HFD-fed, NMN-treated mice in contextual and cued fearconditioning tests on Day 1, Day 2 and Day 3.

FIG. 11 illustrates fundus biomicroscopy images from control andNMN-administered mice.

FIG. 12A-C illustrates electroretinograms from control andNMN-administered mice.

FIG. 13 illustrates tear production in 18 month-old control andNMN-administered mice. All values are presented as mean±SEM. **p<0.01.

FIG. 14A-E illustrates hippocampal NAD+ levels and Nampt expressiondeclining with age.

A) NAD+ biosynthesis from nicotinamide. B) HPLC analysis of NAD+ levelsin hippocampal extracts. C-D) Quantification of immunofluorescence forNampt in the subgranular zone (SGZ). Measurement of thresholded levelsof Nampt immunoreactivity (C) and the number of highly immunoreactiveNampt+ cells (D) along the SGZ. E) Representative images ofimmunofluorescence for Dapi and Nampt in the SGZ in young (6 months old)and old (18 months old) mice.

FIG. 15A-F illustrates that Nampt is expressed in a subpopulation of SGZNSPCs. A-C) Representative fluorescence images for Dapi (original blue),Nampt (original red), and NSPC markers (Sox2, Gfap, and NestinGFP 3 dayspost tamoxifen injection; original green) in the SGZ. Dotted linesdenote the SGZ. D) Quantification of the percentages of NSPCmarker-positive cells in the SGZ that also express Nampt in 3- to6-month old mice.

FIG. 16A-J illustrates that adult NSPC-specific deletion of Namptimpairs NSPC proliferation and self-renewal in vivo. A) To assessproliferation, iNSPC-Nampt-KO mice and littermate controls weresubjected to three rounds of 5 tamoxifen (TAM) injections (1 injectionper day, 6 weeks apart). Sacrifice was performed at 6 months of age. B)A scheme for the specificity of the markers assessed. C-F)Quantification of radial Nestin+ NSPCs (n=15-16 mice) (C), BrdU+proliferating cells (n=14-16 mice) (D), Ki67+ proliferating cells (n=7mice) (E), and newborn neurons (Dcx+, n=15-20 mice) (F), per unit areaof the dentate gyrus (DG) in control and iNSPC-Nampt-KO mice. For BrdUlabeling, 4 injections of BrdU at 100 mg/kg body weight were givenintraperitoneally over 48 hours. G) Representative images ofimmunofluorescence for Gfap (original blue), Dcx (original green), andBrdU (original red) in the subgranular zone (SGZ). Scale bar denotes 200μm. H) To assess differentiation, control littermates and iNSPC-Nampt-KOmice were subjected to 4 total TAM injections (2 injections on the firstday coupled with BrdU at 100 mg/kg body weight as well as 2 totalinjections on the subsequent 2 days). I) Quantification of thepercentage of BrdU+ cells in the DG that also express markers of NSPCs(Gfap+, Nestin+), newborn neurons (Dcx+), and OPCs/oligodendrocytes(Olig2+) (n=6-13 mice). J) Quantification of radial Nestin+ NSPCs in 6and 18 month-old C57B16 mice and 18 month-old C57B16 mice treated with100 or 300 mg/kg body weight NMN in their drinking water for 12 months(n=5 mice). Data are presented as mean±s.e.m. *P<0.05. **P<0.01.***P<0.001.

FIG. 17A-E illustrates that inhibition of Nampt in NSPCs in vitroimpairs NAD+ biosynthesis and proliferation. Neurospheres were culturedwith the Nampt-specific inhibitor FK866 (10 nM) with or without NMN (100μM) for 48 hours. A) HPLC analysis of NAD+ levels (n=6). B)Quantification of the fold increase of cell number in neurospheres(n=6-30). C) Representative bright-field image of neurospheres. Scalebar denote 10 μm. D) Cell cycle-related pathways among the top 50biological pathways downregulated by FK866. Parametric analysis of geneenrichment (PAGE) was conducted based on microarray analyses. See theMethods section. E) Quantitative RT-PCR results for mRNA expression ofcyclin E2 (Cene2), cyclin E11 (Cene1), cyclin A2 (Cena2), and E2F1(n=3). F) FACS analysis of FK866-treated NSPCs (n=8). Data are presentedas mean±s.e.m. *P<0.05. **P<0.01. ***P<0.001.

FIG. 18A-L illustrates that genetic ablation of Nampt in NSPCs in vitroimpairs NAD+ biosynthesis, proliferation, and differentiation.Neurospheres were isolated from Nampt^(flox/flox) mice and infected witha Cre-recombinase expressing adenovirus (Nampt AD-Cre) or a controladenovirus expressing LacZ (Nampt AD-LacZ). A) HPLC analysis of NAD+levels with and without NMN (100 μM, 48 hours) (n=10-22). B-C)Quantification of the fold increase in cell number (n=13-50) andneurosphere diameter (n=9 independent samples, 57-96 neurospheres). D)Representative images of neurospheres 7 days after dissociation. Scalebars denote 10 μm. E) The number of neurospheres formed 7 days afterplating dissociated cells at 100 cells/ml, 0.5 ml/well in 24-well plates(n=8 independent samples, 48-84 wells). F-G) Nampt Ad-Cre and NamptAD-LacZ infected neurospheres were cultured without NMN until NamptAd-Cre infected neurospheres exhibited a growth defect. Cultures werethen passaged and plated at equal density with or without NMN (200 μM).Fold increases in cell number (F) (n=6), and the percentages of totalDapi+ cells that express Ki67+ cells were quantified (G) (n=3independent samples, 9 fields of view). H-L) The percentages of totalDapi+ cells that express the indicated cell type-specific markers (H) byimmunofluorescence after 6-7 days of differentiation: 04 (I), Gfap (J),and B-III-tubulin (K) (n=3-6 independent samples, 23-43 fields of view).The effect of NMN was also examined for O4, S100β, TUNEL, and Nestin (L)(n=3-6 independent samples, 10-26 fields of view). *, {circumflex over( )}, and #indicate statistical significance between Nampt AD-LacZ andNampt AD-Cre, Nampt AD-LacZ and Nampt AD-LacZ+ NMN, and Nampt AD-Cre andNampt AD-Cre+ NMN, respectively. Data are presented as mean±s.e.m.*P<0.05. **P<0.01. ***P<0.001.

FIG. 19A-G illustrates that genetic ablation of Nampt in vitro impairsOPC formation. A) A scheme for oligodendrocyte differentiation withstage-specific markers. B-C) Neurospheres were infected with a Crerecombinase-expressing adenovirus (Nampt AD-Cre) or a control adenovirusexpressing LacZ (Nampt AD-LacZ). To assess oligodendrocyte formation,dissociated neurospheres were harvested after 6-7 days ofdifferentiation (B). To assess OPC formation, dissociated neurosphereswere examined after 2 days of differentiation (C). Markers of NSPCs(Gfap, Nestin), OPCs (Pdgfrα+), and oligodendrocyte lineage cells(Olig2+, O4+) were assessed (n=3-9 independent samples, 6-51 fields ofview). D) Treatment of dissociated neurospheres with the selectiveinhibitor of Sirt1, EX527 (80 μM) or the selective inhibitor of Sirt2,AGK2 (10 μM). The formation of oligodendrocytes was evaluated after 6-7days of differentiation (n=6-11 independent samples, 21-32 fields ofview). E-G) Knockout and control neurospheres were formed by infectingwith a Cre-recombinase expressing adenovirus or a control adenovirusexpressing LacZ, respectively. E) Neurospheres were isolated fromSirt1^(flox/flox) mice and Sirt1^(flox/flox); Sirt2−/− mice. Theformation of oligodendrocytes was evaluated after 6-7 days ofdifferentiation (n=3-11 independent samples, 12-28 fields of view). F-G)Neurospheres were isolated from Nampt^(flox/flox) mice (F, n=8-9) orSirt1^(flox/flox); Sirt2−/− mice (G, n=3-7) and differentiated for 2days. Quantitative RT-PCR results for mRNA expression of oligodendrocytelineage genes. Data are presented as mean±s.e.m. *P<0.05. **P<0.01.***P<0.001.

FIG. 20A-J illustrates adult NSPC-specific deletion of Nampt impairsNSPC self-renewal and differentiation in response to insult-induceddemyelination in vivo. A) Quantification of the percentage ofNestinGFP-positive cells in the SVZ that also express Olig2 in iNSPC-GFP(n=7) and iNSPC-Nampt-KO (n=8) mice 7 days post initial TAM injection.B) 6- to 9-week-old iNSPC-GFP control and iNSPC-Nampt-KO mice were fed adiet containing 0.2% cuprizone for 4-5 weeks. Deletion of Nampt in theadult Nestin+ population was induced by 5 tamoxifen (TAM) injections at180 mg/kg body weight per day the week before starting the cuprizonediet. C) A scheme of a coronal mouse brain section. Original red boxedareas indicate regions used for quantification. Original red dotted lineindicates the SGZ. CC, corpus callosum; DHC, dorsal hippocampalcommissure; DG, dentate gyrus; HPF, hippocampal formation; SCZ,subcallosal zone; SGZ, subgranular zone; V3, third ventricle. D)Quantification of the number of NestinGFP+ cells per unit area in theCC. E) A scheme for the specificity of the markers assessed. F-I)Quantification of the percentages of NestinGFP+ cells that express NSPCmarkers (Nestin, Gfap) or oligodendrocyte markers (Sox10, Ape) in the CC(n=2-11 mice). * and {circumflex over ( )} indicate statisticalsignificance between iNSPC-GFP control littermates and iNSPC-Nampt-KOmice and between regular chow- and cuprizone-fed iNSPC-GFP mice,respectively. J) Representative images of immunofluorescence for Dapi(blue), Nampt (red), and Olig2 (green) in the CC. Arrows indicateexamples of colocalization. Scale bars denote 20 μm. Data are presentedas mean f s.e.m. *P<0.05. **, {circumflex over ( )}{circumflex over( )}<0.01. ***, {circumflex over ( )}{circumflex over ( )}{circumflexover ( )}P<0.001.

FIG. 21 illustrates a model for the role of Nampt-mediated NADbiosynthesis in NSPCs. Nampt-mediated NAD+ biosynthesis promotes NSPCself-renewal, proliferation and differentiation into oligodendrocytes.While the mechanism by which Nampt promotes self-renewal andproliferation remains unidentified, Nampt-mediated NAD+ biosynthesisactivates Sirt1 and Sirt2 to promote NSPC oligodendrocyte lineage fatedecisions by a mechanism involving transcriptional downregulation ofPdgfrα, Sox10, and Nkx2.2 and transcriptional upregulation of p21(cdkn1a). Sirt1 and Sirt2 may act via an effect on Olig2 activity. Seetext for a detailed discussion.

FIG. 22A-1 illustrates Nampt expressed in a subpopulation of SGZ NSPCs.A-C, H-I) Representative images of immunofluorescence for Dapi (blue),Nampt (red), and cell type specific markers (NeuN: mature neurons,S100β: mature astrocytes, Ki67: proliferating cells, Olig2:oligodendrocyte lineage cells; green) in the subgranular zone (SGZ).Dotted lines denote the SGZ. Single arrowheads indicate examples ofcolocalization. Double arrowheads indicate examples ofnon-colocalization. Scale bars denote 10 μm. B) Zoom of boxed regionshown in A). D) A scheme for the specificity of the markers assessed. E)Percentage of Dapi+ cells that express the neuronal marker NeuN in theSGZ (n=5). F) Percentage of Dapi+ cells that express the NSPC markerSox2 in the SGZ (n=5). G) Quantification of the percentages ofmarker-positive cells that also express Nampt in the SGZ (Ki67: n=304cells from 13 mice; Olig2: n=122 cells from 10 mice).

FIG. 23A-I illustrates that adult NSPC-specific deletion of Namptimpairs NSPC proliferation and self-renewal in vivo. A-F) iNSPC-Nampt-KOand littermate control (iNSPC-GFP) mice were injected with tamoxifen(TAM) or vehicle (5 total injections, 1 injection per day). A-B)Representative images of immunofluorescence for Dapi (blue), activatedcaspase 3 (red), and NestinGFP (green) in the indicated brain regions at28 (A) or 3 (B) days post TAM injection. Arrows highlight the rareactivated caspase 3+ cells observed. Scale bars denote 50 μm. A) ControliNSPC-GFP mice were treated with oil or TAM to ensure that there was noleaky NestinGFP reporter expression. B) iNSPC-Nampt-KO or iNSPC-GFP micewere treated with TAM. C) Recombination-confirmatory PCR performed onhippocampal DNA from TAM treated iNSPC-Nampt-KO (KO) and control mice(n=7-8). D) Quantification of the percentages of NestinGFP-positivecells in the SGZ that also express NSPC (Sox2: n=190 cells from 7 mice;Gfap: n=208 cells from 7 mice) or neuronal (Dcx, NeuN, n=473 cells from7 mice) markers in 3 to 6 month old iNSPC-GFP mice 7 days post initialTAM injection. E) Quantification of the percentages ofNestinGFP-positive cells that also express Nampt in iNSPC-Nampt-KO andiNSPC-GFP mice in the DG at the indicated days post initial TAMinjection (n=more than 350 cells from 7 mice). F) Newborn neurons (Dcx+,n=12-16) were categorized by the length of their projection per unitarea of the dentate gyrus (DG). G) Mice were injected with NMN (500mg/kg body weight, IP), and hippocampal NAD+ levels were measured byHPLC at the indicated time points post injection (n=3-9). H-I) Mice wereadministered NMN (100 or 300 mg/kg body weight) in their drinking waterfrom 6 to 18 months of age.

FIG. 24A-E illustrates that inhibition of Nampt in NSPCs impairs NAD+biosynthesis and proliferation in vitro. Neurospheres were cultured withthe Nampt-specific inhibitor FK866 (10 nM) with or without NMN (100 μM)for 24 (A-B) or 48 hours (C-G). A) HPLC analysis of NAD+ levels (n=6).B) Quantification of the fold increase of cell number in neurospheresunder each condition indicated (n=5-11). C) A representative immunoblotof FK866-treated neurospheres. D-E) Quantification of immunoblots forKi67 (D) and Pcna (E) normalized by actin in neurospheres (n=6). F-G)Top 50 biological pathways downregulated (F) or upregulated (G) byFK866.

FIG. 25A-G illustrates genetic ablation of Nampt in NSPCs in vitroimpairs NAD+ biosynthesis, proliferation, and differentiation. A-G)Neurospheres were isolated from Namptflox/flox mice and infected with aCre-recombinase expressing adenovirus (Nampt AD-Cre) or a controladenovirus expressing LacZ (Nampt ADLacZ). A) Quantitative RT-PCRresults for mRNA expression of Nampt in AD-LacZ and Nampt Ad-Creinfected neurospheres (n=3-33). B) Representative immunoblots for Namptand Gapdh. C) Quantification of immunoblots for Nampt in neurospheresnormalized by Gapdh (n=4-13). D) HPLC analysis of NAD+ levels. NAD+levels in Nampt Ad-Cre infected neurospheres were normalized by NAD+levels in Nampt Ad-LacZ infected neurospheres (n=4-9). E) Representativeimmunoblots of Nampt Ad-Cre or Nampt AD-LacZ infected neurospheres 8days post infection for markers of cell death (activated caspase 3) andproliferation (Ki67, Pcna). Neurospheres were grown under proliferationconditions (left blot) or differentiated for 2 days (right blot). F)Immunofluorescence analysis of dissociated neurospheres cultured inproliferation media. Histogram shows the percentages of activatedcaspase 3+(n=3 independent samples, 6 fields of view) or TUNEL+ cells(n=9 independent samples, 14-21 fields of view) relative to the totalnumber of Dapi+ cells. G) A scheme for the non-directed lineagedifferentiation protocol used.

FIG. 26A-J illustrates A) A scheme for the oligodendrocytic lineagedifferentiation protocol used. B) Histogram shows the percentages ofDapi+ cells that express markers of NSPCs (Gfap, Nestin), OPCs (Pdgfrα+,Olig2+), and astrocytes (S100β) (n=3-12 independent samples, 6-30 fieldsof view). C) A representative immunoblot for Sirt2 in neurospherescultured as NSPCs (with EGF, FGF) or OPCs (with EGF, FGF, PDGFαα) beforeand after differentiation. D) immunofluorescence for Dapi (originalblue), Nampt (original red), and Sirt2 (original green) along the SGZ.Dotted lines denote the SGZ. Single arrowheads indicate examples ofcolocalization of cell immunoreactivity. Scale bar denotes 10 μm. E-F)immunofluorescence for Dapi (blue), Sirt2 (red), and NestinGFP (originalgreen, 3 days post TAM) along the SGZ. Dotted lines denote the SGZ. E)Scale bar denotes 50 μm. F) Scale bar denotes 20 μm. G-H) Neurosphereswere isolated from Sirt1 flox/flox mice and infected with a Crerecombinase-expressing adenovirus (Sirt1 AD-Cre) or a control adenovirusexpressing LacZ (Sirt1 AD-LacZ). G) Quantitative RT-PCR results for mRNAexpression of Sirt1 (n=17-24). H-J) Quantification of the fold increasein cell number (n=5-20). Neurospheres were derived from full body Sirt1KO mice (I), Sirt2 KO (J) mice, and their respective littermatecontrols.

FIG. 27A-H illustrates adult NSPC specific deletion of Nampt impairsNSPC self-renewal in response to insult-induced demyelination in vivo.A) Representative images of immunofluorescence for Dapi (blue), Nestin(red), and NestinGFP (green), and in regular chow (RC) and cuprizone fed(CUPR) mice in the indicated regions of the brain: SGZ, subgranularzone; SVZ, subventricular zone; CC, corpus callosum. Scale bars denote20 μm. B) Representative images of immunofluorescence for Dapi (blue),MBP (red), and NestinGFP (green) in regular chow- and cuprizone-fed micebefore and after 1 week of recovery in the SGZ. Scale bars denote 20 μm.C-G) Quantification of the number of NestinGFP+ cells per unit area ofthe dentate gyrus (C) and percentages of NestinGFP+ cells that expressNSPC markers (Nestin, Gfap) or oligodendrocyte markers (Sox10, Ape) inthe SGZ (D-G) (n=5-12 mice). * and {circumflex over ( )} indicatestatistical significance between iNSPC-GFP control littermates andiNSPC-Nampt-KO mice and between regular chow- and cuprizone-fed iNSPCGFPmice, respectively. H) Representative images of immunofluorescence forDapi (blue), Nampt (red), and Sox2 (green) in the CC. Arrows indicateexamples of colocalization of immunoreactivity. Scale bars denote 20 μm.

FIG. 28A-B illustrates the NAD biosynthetic pathway from nicotinamide.(A) The rate-limiting step catalyzed by nicotinamidephosphoribosyltransferase (NAMPT). Nic, nicotinamide; PRPP,5′-phosphoribosyl-1-pyrophosphate; NMN, nicotinamide mononucleotide;PPi, pyrophosphate. (B) The NAD biosynthetic pathway from nicotinamide.

FIG. 29A-L illustrates (A-B) retinas from NAMPT rod-CKO mice showed asignificant reduction of NAMPT within rods by PCR, immunohistochemistryand immunoblotting. (C) Neurosensory retinal degeneration was associatedwith secondary atrophy and pallor of the optic nerve. (D-F)Electroretinography (ERG) was performed to measure PR neuron and retinalfunction. (G) Photopic visual acuity measurements confirmed vision lossin rod-CKO mice. (H) Histopathologic examination of eyes from NAMPTrod-CKO mice. (I) Normalized NAD measurements obtained from NAMPTrod-cko whole retinas. (J-L) CKO mice treated with NMN showedsignificant rescue of photopic and scotopic function

FIG. 30A-L illustrates (A) NAMPT cone-CKO mice (B-D) ERG demonstratedprogressive decline in cone function. (E) Significant decrease in visualacuity in cone-CKO mice. (F-H) ERG function compared to PBS treatedcone-CKO mice. (I-J) FK866 treatment of cone cells in vitro causesdecrease in intracellular NAD levels and significant cell death afterthe 4 hours of treatment. (I-K) NMN (100 μM) was able to completelyrescue cells. (L) depicts histopathologic examination of eyes from NAMPTrod-CKO mice.

FIG. 31A-N illustrates (A-J) Retinal and PR neuron structure andfunction. (K-M) Examination of mice that had normal retinal structureand function.

FIG. 32A-E illustrates (A-D) Electron microscopic examination. (E) Roleof NAD in NAMPT-mediated effects on PR neurons.

FIG. 33A-B illustrates NMN treatment effect on littermate controls.

FIG. 34A-F illustrates retinal and PR neuron structure and function.

FIG. 35 illustrates changes in inner segments could be identified inrod-CKO mice.

DETAILED DESCRIPTION Abbreviations

BMD: Bone mineral density

CC: Corpus callosum

DG: Dentate gyrus

DXA: Dual-energy X-ray absorptiometry

EIR: Enhanced immediate release

ERG: Electroretinography

FFA: Free fatty acid

HFD: High fat diet

NMN: Nicotinamide mononucleotide

OPC: Oligodendrocyte precursor cells

PR: Photoreceptor

SGZ: Subgranular zone

SVZ: Subventricular zone

Methods

The methods and compositions described herein utilize laboratorytechniques well known to skilled artisans, and can be found inlaboratory manuals such as Sambrook, J., et al., Molecular Cloning: ALaboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 2001; Spector, D. L. et al., Cells: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1998; Nagy, A., Manipulating the Mouse Embryo: A Laboratory Manual(Third Edition), Cold Spring Harbor, N.Y., 2003 and Harlow, E., UsingAntibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1999. Methods of administration ofpharmaceuticals and dosage regimes, can be determined according tostandard principles of pharmacology well known skilled artisans, usingmethods provided by standard reference texts such as Remington: theScience and Practice of Pharmacy (Alfonso R. Gennaro ed. 19th ed. 1995);Hardman, J. G., et al., Goodman & Gilman's The Pharmacological Basis ofTherapeutics, Ninth Edition, McGraw-Hill, 1996; and Rowe, R. C., et al.,Handbook of Pharmaceutical Excipients, Fourth Edition, PharmaceuticalPress, 2003. As used in the present description and the appended claims,the singular forms “a”, “an” and “the” are intended to include theplural forms as well, unless the context indicates otherwise.

The following Methods are applicable to Examples 1-7:

Administration of NMN Through Drinking Water and Determination of NMNStability and Toxicity

A 12 month-long NMN administration study using wild-type mice under aregular chow-fed condition was conducted. NMN was administered throughdrinking water, and two doses of NMN, 100 and 300 mg/kg body weight/day,were tested. The stability of NMN was examined in drinking water andfound that NMN was stable in solution. No significant degradation wasobserved at room temperature. Water intake was also monitored verycarefully, and the water intake did not change significantly through theexperimental period.

To assess beneficial and possible adverse effects of NMN, a variety ofphysiological parameters were periodically monitored, including bodyweight, body temperature, food and water intake, fed and fasted bloodglucose levels, fed and fasted plasma lipid panels, and glucose andinsulin tolerance, in NMN-administered and control mice. Bloodchemistry, blood cell counts, urine strip test, and other physiologicaltests including physical activity test were also checked. Based on allthese assessments, no adverse effects, such as malnutrition, or signs oftoxicity were observed in either of the 100 mg/kg or 300 mg/kg groups.

Memory Function Study

Two groups of wild-type C57BL/6 mice at ˜2 months of age were fed a highfat diet (HFD) containing 42% of the total calories from fat (TD88137;Harlan Taklad). NMN at a dose of 300 mg/kg/day began to be administeredthrough drinking water to one of the HFD-fed groups after 4 months ofHFD feeding. The control group was fed a regular chow. After 8 months ofHFD feeding with or without 4 months of NMN treatment, the contextualfear conditioning test, a sensitive test to examine the memory functionthat involves the hippocampus, was conducted for mice in these threegroups.

The following Methods are applicable to Examples 8-15

Mice

Mice were maintained on a regular chow ad libitum on a 12 hr light/darkcycle (lights on from 6 am to 6 μm). Namptflox/flox mice (Rongvaux etal, 2008), in which exons 5 and 6 of the Nampt gene are flanked by loxPsites, were crossed to Nestin-CreERT2 mice (Lagace et al, 2007) togenerate Nampt flox/+; Cre double heterozygous mice. Double heterozygousmice were bred to Namptflox/flox mice to obtain Namptflox/flox; Cremutant mice (iNSPC-Nampt-KO mice) in the expected Mendelian ratio. Totrace the progeny of adult NSPCs and to confirm the specificity andmagnitude of the recombination induced by tamoxifen injection,iNSPC-Nampt-KO and Nestin-CreERT2 mice were crossed to a reporter mousestrain that expresses a loxP-flanked STOP cassette that preventstranscription of the downstream enhanced green fluorescent protein[ZsGreen1; Jackson laboratories #7906 (Madisen et al, 2010)].Recombination PCR on hippocampal extracts of tamoxifen or vehicletreated mice showed successful deletion upon treatment with tamoxifen(FIG. 23C)

Induction of Nampt Deletion

Tamoxifen injections were performed as described previously (Lagace etal, 2007). Briefly, iNSPC-Nampt-KO mice (5-7 weeks old) wereadministered tamoxifen (TAM, Sigma T5648) at 180 mg/kg/d for 5 days (d,intraperitoneally; dissolved in 10% EtOH/90% sunflower oil), a protocolthat produces maximal recombination with minimal lethality (5%) (Lagaceet al, 2007).

BrdU Incorporation

5′-bromodeoxyuridine (BrdU, Sigma, B9285) was diluted in sterile salineand administered by intraperitoneal injections (100 mg/kg body weight).For analysis of the cumulative effects of loss of Nampt, mice were givenBrdU twice a day for 2 days and sacrificed the following day or 28 dayslater. For analysis of the effect of loss of Nampt on adult NSCdifferentiation and postnatal oligodendrocyte differentiation, mice weregiven BrdU twice a day for 1 day and sacrificed 2 days later.

Cuprizone

Demyelination was induced by feeding 6 to 8-week-old mice a dietcontaining 0.2% cuprizone (bis-cyclohexanone oxaldihydrazone; SigmaC9012) mixed into a ground standard rodent chow for 4 to 5 weeks (HarlanLaboratories, TD.01453). To allow recovery from cuprizone treatment,food was replaced with standard chow for an additional 1 week. Thisprotocol has been shown to successfully demyelinate and remyelinate thehippocampus (Skripuletz et al, 2011).

Immunofluorescence

All tissue sections were and cells incubated inblocking/permeabilization solution containing 10% normal goat serum, 1%BSA, and 0.3% Triton-X in PBS for 45 to 60 min prior to 24 or 48 h ofincubation with primary antibodies in 5% normal goat serum and 0.1%Triton-X in PBS at 4° C. at the concentrations listed below. Alexa627,Alexa488, or Cy3 conjugated-secondary antibodies diluted in 2% normalgoat serum, 1% BSA, and 0.1% Triton-X in PBS were added for 2 h at roomtemperature. Nuclei were stained with 4,6-diamidino-2-phenylindole(Sigma) for 10 min at room temperature.

Cells were harvested by fixation with 4% paraformaldehyde in PBS (15min). Mice were anesthetized by i.p. injection of ketamine and xylazine,and perfused transcardially through left ventricle with cold 0.1 Mphosphate buffer at pH 7.4 followed by a phosphate-buffered solution of4% paraformaldehyde (PFA). Brains were postfixed with 4% PFA overnightand placed into 15% sucrose followed by 30% sucrose, frozen, and storedat −80° C. until use. Coronal sections (30 μm) were made by cryostat ina 1 in 8 series and stored at −30° C. in cryoprotectant until use. Toremove any endogenous peroxidase activity, all sections were incubatedwith 3% H2O2 for 10 min. Tissue sections used to assess BrdUincorporation were treated before the immunostaining procedure with 50%formamide in 2× saline/sodium citrate (SSC) at 65° C. for 2 h, 2N HClfor 30 min at 37° C., 0.1 M borate pH 8.5, and then washed twice withPBS before proceeding with the staining protocol. Tissue sections notused to assess BrdU incorporation were either incubated in 50% formamidein 2× saline/sodium citrate (SSC) at 65° C. for 2 h or 10 mM citratebuffer at 65° C. for 1 h before proceeding with the staining protocol.Detection of Dcx, Nestin, Nampt, and APC was performed using theTSA-Plus fluorescein kit (PerkinElmer).

Quantification

For tissue sections, high-magnification (20×, 0.8DICII or 40× oil1.3DICII) microscopic imaging was performed using a Zeiss Axioimage.Z1.Images were taken in z-stacks of 1 μm steps through the range of tissuesection immunoreactivity. For the dorsolateral corner of the SVZ, imageswere taken from bregma 1.10 to −0.10 mm. For the corpus callosum, imageswere taken from bregma −1.06 to −2.54 mm. For the dentate gyrus, imageswere taken from bregma −1.34 to −3.64 mm. Quantification was performedblinded to genotype on 3-8 tissue sections per animal. Cell densitieswere estimated by the number of immunoreactive cells divided by the areaof the structure, measured with ImageJ. Verification of colocalizationwas achieved by importing stacks of Z images into ImageJ and performing3D rendering. For cells, 10 or 20× microscopic imaging was performedusing a Zeiss Axioimage.Z1. Quantification was performed blinded togenotype on 2 to 3 fields of view per sample and treatment, from 3 to 9independent samples.

NAD+ Measurement

NAD+ levels were determined using an HPLC system (Shimadzu) with aSupelco LC-18-T column (15 cm×4.6 cm; Sigma), as described previously(Yoshino et al, 2011).

Microarrays and Bioinformatic Analyses

For individual genes, raw microarray data were subjected to Z scoretransformation, and Z ratios were calculated as described previously(Cheadle et al, 2003). Subsequent analysis and Parametric Analysis ofGene Set Enrichment (PAGE) analysis was performed as previouslydescribed (Yoshino et al, 2011). The microarray data used in this studyhas been deposited into the NCBI GEO database (GEO accession numberGSE49784).

Western Blotting

Protein extracts (15-50 μg) from mouse hippocampi or neurospheres wereprepared as previously described (Yoshino et al, 2011).

Quantitative Real-Time RT-PCR

Total RNA was extracted from the hippocampus using an RNeasy® kit(Qiagen®) and reverse-transcribed into cDNA with a High Capacity cDNAReverse Transcription kit (Applied Biosystems). Quantitative real-timeRT-PCR was conducted with the TaqMan®. Fast Universal PCR Master mix(Applied Biosystems) and appropriate TaqMan® primers for each gene withthe GeneAmp® 7500 (Applied Biosystems) fast sequence detection system.Relative expression levels were calculated for each gene by normalizingto Gapdh levels and then to a control.

Reagents

The following primary and secondary antibodies were used:

Primary antibodies and their uses or cell type specificities (See vonBohlen und Halbach, 2011): Actin: normalization, WB 1:4000 CPO1, Sigma;Gapdh: normalization, WB 1:4000 6C5 Millipore CB1001; Nampt: IHC 1:1000;WB 1:3000 Alexis Biochemicals ALX-804-717-C100; Pdgfrα: oligodendrocyteprecursor cells, IF 1:500 APA5 BD Biosciences; Olig2: alloligodendrocyte lineage cells, IHC 1:500, IF 1:1000; Millipore; O4:immature oligodendrocytes, IF 1:1000 Millipore, MAB345; APC:oligodendrocytes, IHC 1:1000 Millipore CC-1 OP80; MBP: matureoligodendrocytes, IHC 1:1000 Millipore MAB386; Ki67: proliferatingcells, IHC, IF 1:500; WB 1:3000 Abcam ab66155; Pcna: proliferatingcells, WB 1:2000; PC10 Cell signaling #2586; 5-bromo-2′-deoxyuridine(BrdU): a thymine analog that incorporates into the DNA of cells in Sphase, IHC 1:500; OBT0030 Accurate; Activated caspase 3: apoptosis, IHC,IF 1:500; Cell Signaling #9661; LC3B: autophagy, WB 1:1000; NovusNB600-1384; TUNEL: cell death, Roche In Situ Cell Death Detection Kit 11684 795 910; Dcx: newly born neurons, IHC 1:1000; Cell Signaling #4604;NeuN: mature neurons, IHC, 1:500, Millipore, MAB377; Nestin: NSPCs, IHC,IF 1:1000, Millipore MAB353; Sox2: NSPCs, IHC, IF 1:500; WB 1:2000;Millipore AB5603; Gfap: NSPCs and astrocytes, IHC, IF 1:1000; MilliporeMAB360;

Secondary antibodies: Jackson ImmunoResearch anti-rat, anti-rabbit,anti-mouse Cy3 (1:400), Alexa Fluor488® (1:200), and Alexa Fluor647®(1:200) (Life Technologies Corporation). Anti-rabbit, anti-mousehorseradish peroxidase (Invitrogen).

FK866 (Hasmann & Schemainda, 2003) (Sigma F8557), EX527 (Peck et al.,2010) (Cayman Chemical 10009798), and AGK2 (Outeiro et al, 2007) (SigmaA8231) were dissolved in DMSO and used to inhibit Nampt, Sirt1, andSirt2 respectively.

Neurosphere Culture

Neurosphere cultures and culture media were prepared as described byDasgupta & Gutmann, 2005 and Lu & Ramanan, 2012 with the following minormodifications. Briefly, postnatal hippocampi were dissected inHibernate-A® (Invitrogen, A12475-01) and trypsinized at 37° C. for 7 m.Cells were mechanically dissociated by pipetting and pelleted bycentrifugation (1700 rpm, 7 min). Dissociation medium (0.1% sodiumbicarbonate, 15 mM HEPES, 0.5% glucose in HBSS) was used to wash thecells before they were resuspended in growth medium. Growth mediumconsisted of DMEM:F12 (1:1, Invitrogen 11966-025 and 21700-075,respectively), B27 (Invitrogen, 17504-044), N2 (Invitrogen, 17502-048),Pen/Strep (Invitrogen), epidermal growth factor (EGF, 20 ng/ml, Sigma,E4127), fibroblast growth factor (FGF, 10 ng/mL, R&D Systems, 233-fb),and heparin (Sigma). Cultures were maintained at 37° C. with 5% CO2, andpassaged twice before use in experiments. Three to nine independentsamples, each in 1 to 3 replicates, from at least two different litters,were used in all experiments. Neurospheres were cultured in thephysiological glucose level of 5 mM (Dienel & Cruz, 2006), which hasbeen previously shown to have no negative consequences on NSPCproliferation, differentiation, or death (Fu et al, 2006; Gao & Gao,2007).

Neurosphere Infection

Neurospheres derived from Nampt^(flox/flox) mice were infected with Ad5Cre recombinase- or b-galactosidase-expressing (LacZ, control)adenoviruses at an MOI of 100. All assessments were performed at least 6days post infection.

Neurosphere Proliferation Analysis

Neurospheres derived from Nampt^(flox/flox) mice were dissociated bytrypsin digestion and seeded at similar cell densities in 24-well plateswith fresh growth medium. Every 24 hours, neurospheres from triplicatewells were collected, dissociated, and counted on a hemocytometer using0.2% trypan blue exclusion to distinguish viable cells. For analysis ofneurosphere diameter, the largest neurosphere in each well was imaged(20× objective) and the diameter was calculated using ImageJ. Forsecondary neurosphere analysis, the total number of neurospheres in eachwell was counted at 7 days post-plating.

Neurosphere Differentiation

Three to five days after their first passage, neurospheres weretrypsinized, washed with dissociation medium, and plated at 150,000cells per well in 24-well plates in differentiation medium [growthmedium without FGF and EGF and with BDNF (5 ng/mL, Peprotech, 450-02) onglass coverslips coated with poly-D-lysine (50 ug/mL; Sigma) and laminin(20 ug/mL; BD Biosciences)]. 6-well plates were coated withpoly-D-lysine (20 ug/mL) and laminin (10 ug/mL). To enrich foroligodendrocytes, PDGFαα (10 ng/ml, Peprotech 100-13A) was added toneurospheres at passage 2 and PDGFαα (2.5 ng/ml) and3,3-,5-triiodo-L-thyronine (T3, 40 ng/ml, Sigma T4397) were added todifferentiation medium. The percentage of oligodendrocyte precursorcells (OPCs) generated was analyzed after 2 d of differentiation, andthe percentage of differentiated oligodendrocytes was analyzed after 6-7d of differentiation.

Statistical Analyses

Differences between two groups were assessed using the Student'sunpaired t-test. Comparisons among several groups were performed usingone-way ANOVA with the Tukey-Kramer post hoc test except for FIG. 16Jand FIG. 24D-E, in which the Games-Howell post-hoc test and the FisherLSD posthoc test were used, respectively. P values <0.050 wereconsidered statistically significant.

The following Methods are applicable to Example 15

Administration of NMN Through Drinking Water and Determination of NMNStability and Toxicity

A 12 month-long NMN administration study using wild-type mice under aregular chow-fed condition was conducted. NMN was administered throughdrinking water, and two doses of NMN, 100 and 300 mg/kg body weight/day,were tested. The stability of NMN was examined in drinking water andfound that NMN was stable in solution. No significant degradation wasobserved at room temperature. Water intake was also monitored verycarefully, and the water intake did not change significantly through theexperimental period. To assess beneficial and possible adverse effectsof NMN, a variety of physiological parameters were periodicallymonitored, including body weight, body temperature, food and waterintake, fed and fasted blood glucose levels, fed and fasted plasma lipidpanels, and glucose and insulin tolerance, in NMN-administered andcontrol mice. Blood chemistry, blood cell counts, urine strip test, andother physiological tests including physical activity test were alsochecked. Based on all these assessments, no adverse effects, such asmalnutrition, or signs of toxicity were observed in either of the 100mg/kg or 300 mg/kg groups.

EXAMPLES

The present teachings including descriptions provided in the Examplesthat are not intended to limit the scope of any claim or aspect. Unlessspecifically presented in the past tense, an example can be a propheticor an actual example. The following non-limiting examples are providedto further illustrate the present teachings. Those of skill in the art,in light of the present disclosure, will appreciate that many changescan be made in the specific embodiments that are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the present teachings.

Example 1

This example illustrates a suppressive effect of NMN on age-associatedbody weight increase.

In these experiments, NMN was administered to mice at a dosage rate of100 mg/kg per day or 300 mg/kg per day. NMN demonstrated a suppressiveeffect on age-associated body weight increase in a 12 month-long NMNadministration study. (See Methods; Administration of NMN throughDrinking Water and Determination of NMN Stability and Toxicity) In theseexperiments, NMN demonstrated a suppressive effect on age-associatedbody weight increase (FIG. 2). The results were analyzed with two-wayRANOVA and one-way RANOVA with the unweighted linear term. All valuesare presented as mean±SEM (n=15, 14, and 14 for control, 100, and 300mg/kg NMN-administered groups).

Average body weights in each group are shown through 0-12 months. Therewas a statistically highly significant interaction between time andgroup (P<0.001 from the two-way RANOVA), and linear dose-dependenteffects were statistically significant at all time points through 4-12months (P<0.05 from one-way RANOVA with the unweighted linear term). Theaverage percent body weight reduction normalized to control mice were 4%and 9% in 100 and 300 mg/kg groups, respectively.

This suppressive effect of NMN on age-associated body weight increasewas further recognized by calculating body weight gains in each group(FIG. 3 0569) in FIG. 3, average body weight gains in each group areshown through 0-12 months. The results were analyzed with two-way RANOVAand one-way RANOVA with the unweighted linear term. All values arepresented as mean±SEM (n=15, 14, and 14 for control, 100, and 300 mg/kgNMN-administered groups). The interaction between time and group wasstatistically highly significant (P<0.001 from the two-way RANOVA), andthe linear dose-dependent effects were significant at all points through2-12 months (P<0.01 from the one-way RANOVA with the unweighted linearterm). The average numbers of percent body weight gain suppressionnormalized to control mice were 12% and 30% in 100 and 300 mg/kg groups,respectively.

Taken together, these results from this 12 month-long NMN administrationstudy demonstrate that NMN can suppress age-associated body weightincrease in a dose-dependent manner, without showing any serious sideeffects during the entire experimental period. These results demonstratethat NMN can be used for the treatment, reduction or prevention ofage-associated obesity.

Example 2

This example illustrates an enhancement of energy metabolism over agewith NMN administration.

In this long-term NMN administration study (See Methods; Administrationof NMN through Drinking Water and Determination of NMN Stability andToxicity), the inventors measured oxygen consumption, energyexpenditure, and respiratory quotient for control, mice administered 100mg/kg NMN per day and mice administered 300 mg/kg NMN per day at the 12month time point by using the Oxymax Lab Animal Monitoring System(Columbus Instruments, Columbus, Ohio).

FIG. 4 shows oxygen consumption in control, 100 and 300 mg/kgNMN-administered mice. The data were analyzed by Wilcoxon signed rankstest. All values are presented as mean±SEM (n=5 in each group).***P<0.001. As illustrated in FIG. 4, oxygen consumption significantlyincreased in both 100 mg/kg and 300 mg/kg groups when examined at times0 through 27 (P<0.001, Wilcoxon signed ranks test).

Energy expenditure measurements also showed significant increases inboth 100 mg/kg and 300 mg/kg groups through 24 hours (P<0.001, Wilcoxonsigned ranks test) (FIG. 5). Energy expenditure in control, 100 and 300mg/kg NMN-administered mice is presented in FIG. 5. The data wereanalyzed by Wilcoxon signed ranks test. All values are presented asmean±SEM (n=5 in each group). ***P<0.001.

Respiratory quotient in control, 100 and 300 mg/kg NMN-administered miceare presented in FIG. 6. The data were analyzed by Wilcoxon signed rankstest. All values are presented as mean±SEM (n=5 in each group).***P<0.001. Respiratory quotient significantly decreased in both groups(P<0.001, Wilcoxon signed ranks test) (FIG. 6 0569). Without beinglimited by theory, these results suggest that NMN increases energyexpenditure by switching their main energy source from glucose to fattyacids, thereby increasing fatty acid oxidation. Without being limited bytheory, this phenomenon could provide an explanation for the suppressiveeffect of NMN on age-associated body weight increase.

Example 3

This example illustrates a suppressive effect of NMN on age-associatedincreases in blood lipid levels.

Blood levels of cholesterol, triglycerides, and free fatty acids areshown over 12 months in the control and the 100 and 300 mg/kgNMN-administered cohorts in FIG. 7A-C. The results were analyzed withtwo-way RANOVA and one-way RANOVA. All values are presented as mean±SEM(n=25 for each group).

Blood levels of cholesterol, triglycerides, and free fatty acids areshown over 12 months in the control and the 100 and 300 mg/kgNMN-administered cohorts. The results were analyzed with two-way RANOVAand one-way RANOVA. All values are presented as mean±SEM (n=25 for eachgroup).

In the control cohort of a long-term NMN study (See Methods;Administration of NMN through Drinking Water and Determination of NMNStability and Toxicity), blood levels of cholesterol showed steadyincreases over time, whereas blood levels of triglycerides and freefatty acids (FFAs) peaked at the 6-month time point and then decreased.However, in both 100 and 300 mg/kg groups, these age-associatedincreases in cholesterol and free fatty acids tended to be suppressed(FIG. 7A-C). In particular, the interaction between time and group wasstatistically highly significant for FFAs (P=0.003 from the two-wayRANOVA), and the 300 mg/kg group did not show any statisticallysignificant increase over time, whereas the control and the 100 mg/kggroups did show statistically significant increases over 12 months andthe first 6 months, respectively (P<0.05 from tests of within-subjectseffects in the one-way RANOVA). All values are presented as mean±SEM(n=25 for each group). Although the average level of free fatty acids atthe 0-month time point in the 300 mg/kg group was significantly higherthan those in the other two groups, NMN at the dose of 300 mg/kgsuppressed age-associated increases in blood levels of FFAs,particularly at the 6-month time point (P<0.05 from the one-way ANOVAwith the Dunnett T3 post-hoc test). Therefore, NMN is capable ofsuppressing age-associated increases in blood lipid levels, particularlyblood FFA levels.

Without being limited by theory, since NMN has an effect of suppressingage-associated body weight increase, it was hypothesized that NMN'seffect on blood lipid levels could be due to the reduction in bodyweight. To address this possibility, lipid levels were compared amongindividual mice whose average body weights were matched through controland experimental cohorts. Body weight-matched blood levels ofcholesterol, triglycerides, and free fatty acids are shown over 12months in the control and the 100 and 300 mg/kg NMN-administered cohortsin FIG. 8A-C. The results were analyzed with two-way RANOVA and one-wayRANOVA. All values are presented as mean±SEM (n=10-15 for each group).

After matching body weight, blood cholesterol levels became very similarthrough control and experimental groups, whereas FFA levels were stilllower in 100 and 300 mg/kg groups compared to those in the control group(FIG. 8A-C). Even after body weight match, the interaction between timeand group was still statistically highly significant for FFAs (P=0.007from the two-way RANOVA), and again, the 300 mg/kg group did not showany statistically significant increases over time, whereas the controland the 100 mg/kg groups showed significant increases over time (P<0.01from tests of within-subjects effects in the one-way RANOVA). Blood FFAlevels tended to be lower in the 100 mg/kg and 300 mg/kg groups comparedto those in the control group after the 6-month time point, although thedifferences did not reach statistical significance (FIG. 8C). All valuesare presented as mean±SEM (n=10-15 for each group). These findingsindicate that NMN has the effect of suppressing the age-associatedincrease in blood FFA levels, independent of the reduction in bodyweight. Without being limited by theory, the effect of NMN on bloodcholesterol levels may be secondary to the effect of suppressingage-associated body weight increase.

It has been reported that chronic treatment with nicotinic acid tends toincrease blood FFA levels, whereas it lowers total cholesterol andtriglyceride levels (Wang, W., et al., Am. J. Phyisol. Endocrinol.Metab. 279, E50-E59, 2000). As shown herein, NMN has a capability ofsuppressing the age-associated increase in blood FFA levels, whichdistinguishes NMN from nicotinic acid. NMN is also able to reducecholesterol levels through the suppression of age-associated body weightincrease. Whereas chronic administration of nicotinic acid can causeskeletal muscle insulin resistance (Fraterrigo, G., et al. Cardiorenal.Med. 2, 211-217, 2012), NMN does not show any adverse effect on glucosemetabolism. Therefore, NMN administration can be an effectiveintervention to suppress age-associated increases in blood lipid levels.

Example 4

This example illustrates that administration of NMN enhances insulinsensitivity in old individuals.

In these investigations, insulin sensitivity, assessed by the insulintolerance test, showed significant differences among the control and the100 mg/kg and 300 mg/kg NMN-administered groups after the 12-month timepoint. (See Methods; Administration of NMN through Drinking Water andDetermination of NMN Stability and Toxicity). As illustrated in FIG.9A-B, insulin tolerance results from body weight-matched mice in thecontrol and the 100 and 300 mg/kg NMN-administered groups at the12-month time point are presented. Blood glucose levels (FIG. 9A) andpercent glucose changes (FIG. 9B) after insulin injection are shown. Theresults were analyzed with two-way RANOVA and one-way ANOVA. All valuesare presented as mean±SEM (n=10-15 for each group).

In these experiments, the last measured time point when mice reached 17month-old, the NMN-administered, body weight-matched mice showedsignificantly enhanced insulin sensitivity compared to the bodyweight-matched control group (FIG. 9A). There was a statisticallysignificant interaction between time and group (P=0.023 from theGreenhouse-Geisser test in two-way RANOVA), and the lineardose-dependent effects were statistically significant or close tosignificance at the 30-min and 45-min time points, respectively (P=0.026and P=0.061 in the one-way ANOVA with unweighted linear term). Theresults were analyzed with two-way RANOVA and one-way ANOVA. All valuesare presented as mean±SEM (n=10-15 for each group). This enhancedinsulin sensitivity in the 100 mg/kg and 300 mg/kg groups was recognizedfurther when plotting percent glucose changes (FIG. 9B), although theinteraction between time and group did not reach statisticalsignificance in this assessment (P=0.091 from the Greenhouse-Geissertest in two-way RANOVA). Long-term NMN administration can enhanceinsulin sensitivity in old mice, indicating that NMN administration isan effective anti-aging intervention to maintain better insulinsensitivity in the elderly.

Example 5

This example illustrates improvement of memory function under a high-fatdiet (HFD) by administration of NMN. (See Methods; Memory FunctionStudy)

FIG. 10 illustrates freezing responses of regular chow-fed control,HFD-fed, and HFD-fed, NMN-treated mice in contextual and cued fearconditioning tests on Day 1, Day 2 and Day 3. NMN was administered atthe dose of 300 mg/kg/day for 4 months. On Day 1 of the study, mice weregiven an auditory cue and then a mild electric foot shock, and a time offreezing was analyzed individually. On Day 2, the trained mice wereplaced into a training chamber with no tone cues, and their freezingresponses were evaluated. On Day 3, cued fear conditioning, which doesnot involve the hippocampus, was tested by giving the same tone cue usedin the conditioning session (Day 1) and analyzing their freezingresponses. HFD-fed mice showed an impairment of contextual fearconditioning on Day 2 compared to regular chow-fed control mice but didnot show any defect in the conditioning session on Day 1 and the cuedfear conditioning test on Day 3 (FIG. 10), demonstrating that HFDspecifically impairs the hippocampus-dependent memory function.NMN-treated, HFD-fed mice demonstrated freezing responsesindistinguishable from those of regular chow-fed control mice in thecontextual fear conditioning test on Day 2. Their freezing responses didnot differ from those of control mice on both Day 1 and Day 3. Theseresults demonstrate that the 4-month administration of NMN in mice canrestore the normal hippocampus-dependent memory function even under aHFD-fed condition. NMN was administered at the dose of 300 mg/kg/day for4 months. The results were analyzed by one-way ANOVA with unweightedquadratic term. All values are presented as mean±SEM (n=17, 15, and 12for regular chow-fed control, HFD-fed, and HFD-fed, NMN-treated mice,respectively). ** P<0.01; * P<0.05. These results demonstrate that the4-month administration of NMN in mice can restore the normalhippocampus-dependent memory function even under a HFD-fed condition.

Example 6

This example illustrates the improvement of retinal photoreceptor cellfunction over age.

In the long-term NMN administration study (See Methods; Administrationof NMN through Drinking Water and Determination of NMN Stability andToxicity), retinal function was evaluated by fundus biomicroscopy andelectroretinography (ERG). FIG. 11 illustrates fundus biomicroscopyimages from control and NMN-administered mice. For each group, five micewere examined, and two representative images are shown.

Intraretinal whitish deposits were reduced dramatically inNMN-administered mice. On fundus biomicroscopy, all five control mice at18 months of age showed many intraretinal whitish deposits, whereas twoand four each out of five mice at 100 mg/kg and 300 mg/kg doses,respectively, showed dramatic reductions in these deposits, suggestingthat age-associated pathological changes in the retina are suppressed byNMN (FIG. 11).

FIG. 12A-C illustrates electroretinograms from control andNMN-administered mice. Amplitudes of scotopic a and b (FIG. 12A, FIG.12B) and photopic b (FIG. 12C) waves over each stimulus range are shown.Consistently, in the ERG analysis, there was a significant interactionbetween stimulus and group (P=0.009 from the two-way RANOVA) for thescotopic a wave, and NMN-administered mice showed significantly higheramplitudes at 0 and 5 db (P=0.035 and 0.022 for the 300 mg/kg group at 0and 5 db, respectively; P=0.009 for the 100 mg/kg group at 5 db from theDunnett T3 test in the one-way RANOVA within groups), demonstrating thatNMN is able to improve rod cell function in aged mice (FIG. 12A). Thedata were analyzed with the two-way repeated ANOVA. All values arepresented as mean±SEM. *p<0.05; p<0.01.

Although there were no significant interactions between stimulus andgroup for the scotopic b and photopic b waves, there appeared to be atrend of improvement for the photopic b wave, which represents cone cellfunction, through an entire range of stimulus in both 100 and 300 mg/kggroups (FIGS. 12B and 12C). The data were analyzed with the two-wayrepeated ANOVA. All values are presented as mean±SEM. *p<0.05; p<0.01.Taken together, these findings indicate that NMN is able to improve theretinal photoreceptor cell function in aged mice.

The present inventors assessed the physiological importance ofNAMPT-mediated NAD biosynthesis in the retina by generating rod cell-and cone cell-specific NAMPT knockout mice. On fundus biomicroscopy,cone cell-specific NAMPT knockout mice had an atrophic appearance at theoptic nerve head, intraretinal whitish deposits, and perivascularsheathing, while littermate control animals were normal. ERGdemonstrated a significant and dramatic decrease in the scotopic b andphotopic b wave amplitudes as compared to the littermate control mice.The scotopic a wave amplitudes in the cone cell-specific NAMPT knockoutmice were significantly decreased but to a lesser extent than thephotopic b wave responses. Furthermore, rod cell-specific NAMPT knockoutmice exhibited total retinal degeneration. Both a and b wave ERGresponses were completely depressed as characterized by a total lack ofresponse to all stimuli. Additionally, the treatment of the mousephotoreceptor-derived 661W cone cell line with FK866, a potent NAMPTinhibitor, led them to apoptotic cell death. Adding NMN to the culturemedia successfully rescued 661W cells from FK866-mediated cell death,suggesting that NAD deficiency causes the observed cell death. Theseresults indicate that inhibition of NAMPT-mediated NAD biosynthesis bygenetic and pharmacologic means leads to photoreceptor cell death andeventually retinal degeneration. NMN administration is an effectiveintervention to treat/prevent retinal degeneration.

Example 7

This example illustrates the improvement of tear production over ageusing modified Schirmer testing.

In this long-term NMN administration study (See Methods; Administrationof NMN through Drinking Water and Determination of NMN Stability andToxicity), tear production was assessed in control and NMN-administeredmice with modified Schirmer's test. All values are presented asmean±SEM. **p<0.01. NMN increased tear production in a dose-dependentmanner in 18 month-old mice (FIG. 13). The tear production observed inthe 300 mg/kg group was comparable to the maximal tear productionthrough the mouse lifespan. These findings indicate that NMNadministration is able to increase tear production significantly in agedmice, providing an effective intervention to protect eye function fromdry eye diseases.

Example 8

This example illustrates that hippocampal NAD+ levels and Namptexpression decline with age.

The inventors hypothesized that aging may reduce Nampt-mediated NAD+biosynthesis in the brain, particularly in the hippocampus, affectingthe function of NSPCs. The inventors first measured NAD+ levels inhippocampi isolated from 1, 3-4, 6, and 10-12 month-old C57B16 mice.FIG. 14A-E illustrates hippocampal NAD+ levels and Nampt expressiondeclining with age. FIG. 14A illustrates NAD+ biosynthesis fromnicotinamide. Nicotinamide phosphoribosyltransferase (Nampt) convertsnicotinamide and 5′-phosphoribosyl-1-pyrophosphate (PRPP) tonicotinamide mononucleotide (NMN). Nicotinamide/nicotinic acidmononucleotide adenylyltransferase (NMNAT) converts NMN andadenosine-5′-triphosphate (ATP) to NAD+. While NAD+ is commonly used inredox reactions, cells primarily require NAD+ as a co-substrate forseveral families of enzymes, one of which is the sirtuin family ofprotein deacetylases. The sirtuin family includes Sirt1 and Sirt2, whichcleave NAD+ at its glycosidic bond, releasing ADP-ribose (Stein & Imai,2012). Inhibitors used in subsequent experiments is indicated. FIG. 14Billustrates HPLC analysis of NAD+ levels in hippocampal extracts (1month, n=5; 3-4 months, n=16; 6 months, n=10; 10-12 months, n=28). C-D)Quantification of immunofluorescence for Nampt in the subgranular zone(SGZ). Measurement of thresholded levels of Nampt immunoreactivity (FIG.14C) and the number of highly immunoreactive Nampt+ cells (FIG. 14D)along the SGZ (n=5). FIG. 14E shows representative images ofimmunofluorescence for Dapi (original blue) and Nampt (original red) inthe SGZ in young (6 months old) and old (18 months old) mice. Scale barsdenote 20 μm. Data are presented as mean±s.e.m. *P<0.05. **P<0.01.***P<0.001.

NAD+ levels gradually decreased with age, reaching 63% in 10-12month-old mice compared to that of 1 month-old mice (FIG. 14B).Consistent with this finding, quantifying Nampt immunoreactivity in theSGZ of the DG by both a thresholded level of Nampt intensity as well asa count of the number of thresholded Nampt+ cells demonstrated that 18month-old mice exhibit 52-66% of the Nampt immunoreactivity present in 6month-old mice (FIG. 14C-E). Data are presented as mean±s.e.m. *P<0.05.**P<0.01. ***P<0.001. Without being limited by theory, these resultssuggest that Nampt-mediated NAD+ biosynthesis in the hippocampusdeclines with age at a time course similar to that of NSPCproliferation.

Example 9

This example illustrates that Nampt is expressed in a subpopulation ofSGZ NSPCs.

Nampt has been reported as predominantly expressed in hippocampalneurons but not in stellate astrocytes (Wang et al, 2011a; Zhang et al,2010). Consistent with this finding, immunohistochemistry for Nampt andcell type specific markers revealed almost all NeuN+ neurons in thegranule layer of the DG expressed Nampt, while almost no S100β+ glialcells did (FIG. 22A-E).

However, the inventors also noticed that many Nampt immunoreactive cellsalong the SGZ of the DG did not express NeuN (FIG. 22B-22E). Theinventors performed co-immunohistochemistry for NSPC markers (Sox2+,radial Gfap+), and found that a significant population of NSPCsexpressed Nampt (FIG. 22A-B 0). FIG. 22 illustrates that Nampt isexpressed in a subpopulation of SGZ NSPCs. A-C) Representative images ofimmunofluorescence for Dapi (original blue), Nampt (original red), andNSPC markers (Sox2, Gfap, and NestinGFP 3 days post tamoxifen injection;original green) in the SGZ. Dotted lines denote the SGZ. Single arrowsindicate examples of colocalization. Double arrows indicate examples ofnon-colocalization. Scale bars denote 10 μm. D) Quantification of thepercentages of NSPC marker-positive cells in the SGZ that also expressNampt in 3 to 6 month old mice. At least 350 cells from 7-14 mice wereassessed per group. E) A representative immunoblot and quantification ofimmunoblots for Nampt normalized by actin in neurospheres cultured frompostnatal mice (n=6 independent samples, 16 replicates), as well ashippocampal tissue extracts (HC) isolated from either postnatal (n=12)or adult mice (n=12). F) Nampt immunoreactivity was thresholded and thenumber of highly immunoreactive Nampt+ cells along the SGZ was assessedfor colocalization with the neuronal marker NeuN or the NSPC marker Sox2in the subgranular zone (SGZ, n=5). Data are presented as mean±s.e.m.*P<0.05. **P<0.01. ***P<0.001 (FIG. 15A-B).

To assess in vivo colocalization between Nampt and Nestin, the inventorscrossed mice expressing Cre recombinase under the Nestin promoter(Nestin-CreERT2) to a GFP reporter mouse strain that expresses aloxP-flanked STOP cassette that prevents transcription of the downstreamenhanced GFP (see Methods), generating iNSPC-GFP mice. Nampt alsocolocalized with GFP driven by the Nestin promoter (NestinGFP, FIG.15C).

Quantification of these observations revealed that along the SGZ, 32% ofSox2+ cells, 55% of radial Gfap+ cells, and 78% of NestinGFP+ cellsexpressed Nampt (FIG. 15D, and table 1). Additionally, Ki67+ and Olig2+cells along the SGZ also expressed Nampt (table 2, FIG. 22H-221).Separate from SGZ-localized cell populations, 3±1% of NestinGFP+ cellshad extremely strong GFP expression, were localized to the granulelayer, and expressed NeuN, likely due to residual CreERT2 protein leftin the progeny of previously differentiated NSPCs. Data are presented asmean±s.e.m. *P<0.05. ***P<0.001.

TABLE 1 Top 50 Downregulated Pathways Pathway Members Changed Z Ratio PValue NUCLEAR_PART 579 468 −7.07 0 DNA_METABOLIC_PROCESS 257 226 −6.49 0DNA_REPLICATION 102 85 −6.2 0 DNA_DEPENDENT_DNA_REPLICATION 56 46 −6.070 DNA_REPAIR 125 111 −5.98 0 NUCLEUS 1433 1169 −5.95 0INTRACELLULAR_ORGANELLE_PART 1192 988 −5.85 0RESPONSE_TO_DNA_DAMAGE_STIMULUS 162 138 −5.8 0 ORGANELLE_PART 1197 992−5.72 0 NUCLEOBASE_NUCLEOSIDE_NUCLEOTIDE_AND_NUCLEIC_ACID 1246 1051−5.34 0 ORGANELLE_LUMEN 458 372 −5.04 0 MEMBRANE_ENCLOSED_LUMEN 458 372−5.04 0 CHROMOSOME 124 106 −4.75 0 CELL_CYCLE_PROCESS 193 167 −4.75 0NUCLEAR_LUMEN 387 310 −4.74 0 CELL_CYCLE_GO_0007049 315 268 −4.63 0RNA_PROCESSING 174 145 −4.32 0 BASE_EXCISION_REPAIR 17 15 −4.3 0CELL_CYCLE_PHASE 170 147 −4.23 0 RESPONSE_TO_ENDOGENOUS_STIMULUS 200 168−4.18 0 MACROMOLECULAR_COMPLEX 945 793 −4.14 0 NUCLEAR_CHROMOSOME 54 45−4.11 0 STRUCTURE_SPECIFIC_DNA_BINDING 56 45 −4.08 0DOUBLE_STRANDED_DNA_BINDING 32 26 −3.95 0CELL_CYCLE_CHECKPOINT_GO_0000075 48 38 −3.92 0 TRNA_METABOLIC_PROCESS 1918 −3.76 0 DNA_RECOMBINATION 47 45 −3.69 0 M_PHASE 114 100 −3.67 0HYDROLASE_ACTIVITY_HYDROLYZING_N_GLYCOSYL_COMPOUNDS 10 10 −3.63 0NUCLEOPLASM 279 230 −3.62 0 REPLICATION_FORK 18 16 −3.6 0CONDENSED_CHROMOSOME 34 27 −3.57 0 TRNA_PROCESSING 10 9 −3.51 0RIBONUCLEOPROTEIN_COMPLEX 143 116 −3.49 0 MITOTIC_CELL_CYCLE 153 133−3.48 0.001 CHROMOSOMAL_PART 96 83 −3.48 0.001 NUCLEOLUS 126 97 −3.470.001 NON_MEMBRANE_BOUND_ORGANELLE 632 513 −3.46 0.001INTRACELLULAR_NON_MEMBRANE_BOUND_ORGANELLE 632 513 −3.46 0.001RNA_BINDING 259 211 −3.44 0.001TRANSFERASE_ACTIVITY_TRANSFERRING_ONE_CARBON_GROUPS 37 34 −3.37 0.001CONDENSED_NUCLEAR_CHROMOSOME 18 15 −3.36 0.001NUCLEOBASE_NUCLEOSIDE_AND_NUCLEOTIDE_METABOLIC_PROCESS 52 45 −3.35 0.001METHYLTRANSFERASE_ACTIVITY 36 33 −3.3 0.001 INTERPHASE 68 57 −3.28 0.001NUCLEOTIDE_METABOLIC_PROCESS 42 36 −3.21 0.001 DNA_INTEGRITY_CHECKPOINT24 16 −3.2 0.001 SINGLE_STRANDED_DNA_BINDING 35 27 −3.17 0.002PROTEIN_COMPLEX 816 689 −3.08 0.002 INTERPHASE_OF_MITOTIC_CELL_CYCLE 6252 −3.07 0.002

TABLE 2 Top 50 Upregulated Pathways Pathway Members Changed Z Ratio PValue SYSTEM_DEVELOPMENT 863 759 4.69 0.000MULTICELLULAR_ORGANISMAL_DEVELOPMENT 1051 910 4.64 0.000SIALYLTRANSFERASE_ACTIVITY 10 10 1.61 0.000 PHOSPHOINOSITIDE_BINDING 2013 4.49 0.000 ANATOMICAL_STRUCTURE_DEVELOPMENT 1017 891 4.47 0.000ORGAN_DEVELOPMENT 572 499 4.28 0.000 RECEPTOR_BINDING 378 321 4.25 0.000EXTRACELLULAR_REGION 448 374 4.10 0.000ENZYME_LINKED_RECEPTOR_PROTEIN_SIGNALING_PATHWAY 140 126 4.05 0.000Growth 77 63 3.85 0.000 NEGATIVE_REGULATION_OF_GROWTH 40 34 3.84 0.000FOCAL_ADHESION_FORMATION 10 9 3.83 0.000OLIGOSACCHARIDE_METABOLIC_PROCESS 11 11 3.82 0.000EXTRACELLULAR_REGION_PART 339 283 3.82 0.000REGULATION_OF_SIGNAL_TRANSDUCTION 223 189 3.77 0.000 SYSTEM_PROCESS 563489 3.74 0.000 EXTRACELLULAR_SPACE 246 204 3.66 0.000 NEURON_PROJECTION21 18 3.62 0.000 INTERMEDIATE_FILAMENT_CYTOSKELETON 24 22 3.61 0.000INTERMEDIATE_FILAMENT 24 22 3.61 0.000 SENSORY_PERCEPTION 190 159 3.570.000 MEMBRANE 1998 1678 3.55 0.000 POSITIVE_REGULATION_OF_SECRETION 2015 3.51 0.000 REGULATION_OF_CELL_GROWTH 46 38 3.49 0.000REGULATION_OF_GROWTH 58 49 3.48 0.000 SIGNAL_TRANSDUCTION 1637 1394 3.470.001 PLASMA_MEMBRANE 1429 1199 3.46 0.001 MESODERM_DEVELOPMENT 22 173.45 0.001 CELL_DEVELOPMENT 579 512 3.44 0.001 FOCAL_ADHESION 13 10 3.440.001 ANATOMICAL_STRUCTURE_MORPHOGENESIS 379 335 3.39 0.001NERVOUS_SYSTEM_DEVELOPMENT 386 342 3.39 0.001 EARLY_ENDOSOME 18 15 3.370.001 CYTOSKELETAL_PROTEIN_BINDING 159 137 3.36 0.001TASTE_RECEPTOR_ACTIVITY 15 3 3.36 0.001 AXON_GUIDANCE 22 19 3.34 0.001GENERATION_OF_NEURONS 83 76 3.34 0.001 CELL_JUNCTION 83 67 3.29 0.001LIPID_HOMEOSTASIS 16 12 3.28 0.001 PDZ_DOMAIN_BINDING 14 13 3.26 0.001LIGAND_DEPENDENT_NUCLEAR_RECEPTOR_ACTIVITY 25 23 3.26 0.001 NEUROGENESIS93 84 3.21 0.001 CELL_MATRIX_JUNCTION 18 14 3.20 0.001 VACUOLE 69 563.19 0.001 IDENTICAL_PROTEIN_BINDING 305 253 3.19 0.001CELL_MATRIX_ADHESION 38 34 3.18 0.001TRANSMEMBRANE_RECEPTOR_PROTEIN_TYROSINE_KINASE_SIGNALING_PATH 83 75 3.180.001 ACTIN_FILAMENT_BASED_PROCESS 116 96 3.18 0.001CELL_SURFACE_RECEPTOR_LINKED_SIGNAL_TRANSDUCTION_GO_000166 642 547 3.170.002 CELL_SUBSTRATE_ADHERENS_JUNCTION 16 13 3.17 0.002

To confirm that Nampt is highly expressed in NSPCs, the inventorscultured NSPCs from the hippocampi of postnatal pups as neurospheres.Neurospheres showed 22 or 32% higher expression levels of Nampt than didwhole hippocampal extracts taken from postnatal (P12) or adult mice(2.5-4.5 months), respectively (FIG. 15E), indicating that NSPCs havehigher expression levels of Nampt compared to other hippocampal celltypes. Without being limited by theory, these results suggest that Namptis expressed in a large subpopulation of NSPCs.

The inventors thresholded Nampt immunoreactivity, and assessed thethresholded Nampt+ cells for colocalization with the neuronal markerNeuN and the NSPC marker Sox2 to determine which cell populations loseNampt expression with age. With age, the percentage of intensely Namptimmunoreactive cells that colocalized with NeuN increased slightly,whereas the percentage of intensely Nampt immunoreactive cells thatcolocalized with Sox2 decreased from 21% to 4% (FIG. 15F). Data arepresented as mean±s.e.m. *P<0.05. **P<0.01. ***P<0.001. Similarly, inthe SGZ, the percentage of NeuN+ that expressed Nampt increased withage, while the percentage of Sox2+ cells that expressed Nampt decreased(FIG. 22E, table 2). Thus, at least part of the decrease in Namptexpression in the SGZ with age is due to loss of Sox2+ NSPCs.

Example 10

This example illustrates that adult NSPC-specific deletion of Namptimpairs NSPC self-renewal in vivo.

The inventors investigated whether inactivating Nampt specifically inadult NSPCs could recapitulate age-associated phenotypic changes in NSPCfunctionality in vivo. The inventors generated adult NSPC-specificinducible Nampt knockout mice by crossing Nampt^(flox/flox) mice(Rongvaux et al., 2008) with Nestin-CreERT2 mice (iNSPC-Nampt-KO mice).To trace the progeny of adult NSPCs in which Nampt was inactivated andto confirm the specificity and magnitude of the deletion induced bytamoxifen, the inventors also crossed iNSPC-Nampt-KO mice to theaforementioned iNSPC-GFP mice. After tamoxifen injection, these miceexpressed NestinGFP in the SGZ and SVZ but not in non-neurogenic regionsof the brain such as the corpus callosum or cortex FIG. 23A-B).

Inmmunohistochemistry and recombination PCR for NestinGFP confirmed thatthere was undetectable recombination present in vehicle injected mice.The ˜350 base pair band confirms the deletion of exons 5 and 6. The1,800 base pair band corresponds to a Nampt gene with a full-length exon5 to 6 sequence. (FIGS. 23A, 23C) To verify that the NestinGFP+population consisted of NSPCs, the inventors co-stained for the NSPCmarkers Sox2 and Gfap. 61% of Sox2+ cells and 34% of radial Gfap+ cellsco-expressed NestinGFP 7 days post tamoxifen (FIG. 23D). The inventorsalso verified Nampt deletion efficiency by quantifying the percentage ofNestinGFP+ cells that expressed Nampt 3 and 7 days post tamoxifeninjection. At 3 days post tamoxifen injection, the percentage ofNestinGFP+ cells that expressed Nampt in iNSPC-Nampt-KO mice was 40%less than littermate controls, and at 7 days post tamoxifen injection,the percentage of NestinGFP+ cells that expressed Nampt was reduced by62% (FIG. 23E).

To assess the cumulative effect of loss of Nampt on NSPC proliferation,the inventors deleted Nampt in iNSPC-Nampt-KO mice at 6 weeks of agewith 3 rounds of 5 consecutive days of tamoxifen injections, separatedby 6 weeks (FIG. 16A). Parametric analysis of gene enrichment (PAGE) wasconducted based on microarray analyses. See the Methods section. Dataare presented as mean±s.e.m. *P<0.05. **P<0.01. ***P<0.001. Theinventors then assessed control and iNSPC-Nampt-KO mice for theexpression of lineage specific markers by immunohistochemistry (FIG.16C). In iNSPC-Nampt-KO mice, the inventors found that the Nestin+ NSPCpool was decreased by 49% in the DG (FIG. 16C). Incorporation of BrdUand the population of proliferating cells [Ki67+(von Bohlen und Halbach,2011)] were also decreased by 22% and 35%, respectively (FIG. 16D-16E.Consistent with this defect in the NSPC pool and proliferation, the poolof newborn neurons [doublecortin, Dcx+, (von Bohlen und Halbach, 2011)]was also significantly decreased by 26% (FIG. 16F-16G). In contrast, theinventors did not observe any significant difference in the maturationof newborn neurons (FIG. 23F). Immature cells had no or horizontalprojections. Mature cells had vertical projections spanning the granulecell layer. NSPC/daughter cell survival was accessed by immunostainingfor activated caspase 3. Only rare activated caspase 3+ cells wereobserved in both neurogenic and non-neurogenic regions of the brain(FIG. 23A-23B), and these activated caspase 3+ cells were never observedin GFP+ cells in iNSPC-Nampt-KO DG, without being limited by theory,providing evidence against a potential contribution of cell death to theobserved effects.

To assess the acute effect of loss of Nampt on NSPC fate decisions, theinventors induced deletion of Nampt at 6 weeks of age with 4 totaltamoxifen injections followed by sacrifice 72 hours after the firstinjection (FIG. 16H). To facilitate assessment of differentiation,dividing cells were labeled by injecting the mice with BrdU concurrentlywith the first day of tamoxifen treatment. 4 total TAM injections (2injections on the first day coupled with BrdU at 100 mg/kg body weightas well as 2 total injections on the subsequent 2 days). iNSPC-Nampt-KOmice displayed significantly reduced levels of colocalization of BrdUwith radial Nestin+ cells (FIG. 16I), without being limited by theory,suggesting decreased self-renewal decisions. However, iNSPC-Nampt-KOmice exhibited normal levels of BrdU colocalization with neuronal(Dcx+), astrocytic (Gfap+) and oligodendrocytic (Olig2+) markers,indicating that alterations in differentiated cell lineage decisionswere undetectable under basal conditions. Without being limited bytheory, the lack of increase in colocalization of BrdU with cell typespecific markers may imply that a larger percentage of BrdU+ cells havefailed to differentiate in iNSPC-Nampt-KO mice. iNSPC-Nampt-KO NSPCscould have stalled during differentiation after losing Nestinexpression.

The inventors hypothesized that systemic administration of NMN may beable to correct age-associated defects in NSPC functionality.Intraperitoneal injection of NMN (500 mg/kg body weight) increasedhippocampal NAD+ levels 34 to 39% within 15 minutes, suggesting that NMNcan cross the blood-brain barrier (FIG. 23G). To see if NMNsupplementation can maintain NSPC proliferation and self-renewal withage, the inventors treated 6 month-old mice with NMN at the daily doseof 100 or 300 mg/kg body weight in their drinking water until 18 monthsof age. The number of Nestin+ cells along the SGZ was significantlylower in the 18 month-old control mice relative to 6 month-old mice, aspreviously reported (Encinas et al, 2011) (FIG. 16J). Data are presentedas mean±s.e.m. *P<0.05. **P<0.01. ***P<0.001. Mice treated with 300mg/kg body weight NMN showed improved maintenance of the Type 1 (radialNestin+) population with age. However, the population of proliferatingcells (Ki67+) remained similar to controls (FIG. 23H). The population ofnewborn neurons (Dcx+) trended to increase (FIG. 23I). Quantification ofKi67+(H) and Dcx+(I) cells in the DG per unit area of the DG (n=5). Dataare presented as mean±s.e.m. *P<0.05. **P<0.01. ***P<0.001. Withoutbeing limited by theory, it is possible that NMN administrationmaintains the NSPC pool by preventing the age-associated increase interminal fate decisions.

Example 11

This example illustrates that inhibition of Nampt in NSPCs in vitroimpairs NAD+ biosynthesis and proliferation.

The inventors hypothesized whether Nampt mediates NSPC-specific NAD+biosynthesis by using hippocampal neurospheres as the in vitro NSPCculture model. Neurospheres were treated with a highly specific Namptinhibitor, FK866, at a dosage and duration (10 nM, 48 hours) that haslittle to no effect on cellular viability (Hasmann & Schemainda, 2003).FK866 reduced NAD+ levels in neurospheres to 4% of controls, a decreasecompletely rescued by concurrent NMN treatment (FIGS. 17A and 24A),suggesting that, without being limited by theory, Nampt activity is thepredominant source of NAD+ biosynthesis in NSPCs.

The inventors investigated how inhibition of Nampt affects neurosphereproliferation. Consistent with the decreases in the NSPC pool and inNSPC proliferation in iNSPC-Nampt-KO mice, FK866 reduced NSPC number by61% after 48 hours, but not 24 hours, of treatment (FIG. 17B-C and FIG.24B). To distinguish whether this decrease in cell number was due to aninhibition of proliferation or enhancement of death, the inventorsanalyzed the protein levels of markers of proliferation, apoptosis, andautophagy. Expression of the proliferation markers Ki67 and PCNAdecreased 87% and 43% respectively (FIG. 24C-24E), whereas levels ofactivated caspase 3 were only slightly increased and levels of theautophagy marker, glycosylated LC3B, were unchanged. Consistent withthese observations, parametric analysis of gene set enrichment (PAGE) ofa microarray performed on neurospheres treated with FK866 showed thatout of the top 50 downregulated pathways, 13 of them were related to thecell cycle, while none of the top 50 upregulated pathways were involvedin cell death table 3, FIGS. 24F-G). Parametric analysis of geneenrichment (PAGE) was conducted based on microarray analyses. See theMethods section. Analysis of specific gene changes by qRT-PCR revealedthat cyclins E and A, the two cyclins required for cellular progressionfrom G1 to S, as well as their upstream transcriptional regulator E2F1(Wong et al, 2011), were the primary cell cycle factors affected by thistreatment (FIG. 17E). These alterations in gene expression indicatedthat reducing Nampt activity stalls NSPCs at G0/G1. Supporting thisnotion, FACS analysis of neurospheres demonstrated that FK866 treatmentincreased the proportion of NSPCs in G0/G1 and decreased the proportionin S phase (FIG. 17F). Data are presented as mean±s.e.m. *P<0.05.**P<0.01. ***P<0.001.

TABLE 3 Impli- Z Ra- P Rank Cell Cycle Total cated tion Value 3DNA_REPLICATION 102 85 −6.20 0.000 4 DNA_DEPEN- 56 46 −6.07 0.000DENT_DNA_REPLICATION 14 CELL_CYCLE_PROCESS 193 167 −4.75 0.000 15NUCLEAR_LUMEN 387 310 −4.74 0.000 16 CELL_CYCLE_GO_0007049 315 268 −4.630.000 19 CELL_CYCLE_PHASE 170 147 −4.23 0.000 25 CELL_CYCLE_CHECK- 48 38−3.92 0.000 POINT_GO_0000075 28 M_PHASE 114 100 −3.67 0.000 31REPLICATION_FORK 18 16 −3.60 0.000 32 CONDENSED_CHROMOSOME 34 27 −3.570.000 35 MITOTIC_CELL_CYCLE 153 133 −3.48 0.001 45 INTERPHASE 68 57−3.28 0.001 50 INTERPHASE_OF_MI- 62 52 −3.07 0.002 TOTIC_CELL_CYCLE

Example 12

This example illustrates that genetic ablation of Nampt in NSPCs invitro impairs NAD+ biosynthesis, proliferation, and differentiation.

To assess the effect of chronic Nampt ablation on NSPC functionality,the inventors genetically ablated Nampt by infecting neurospheres fromNampt^(flox/flox) mice with Cre recombinase- or LacZ-expressing(control) adenoviruses. Neurospheres infected with Cre recombinase(Nampt Ad-Cre) at passage 1 exhibited a 94% reduction in Nampt mRNAexpression 3 days post deletion, and the corresponding decreases inNampt protein expression and NAD+ levels appeared 6 days post deletion(FIGS. 25A-25E). Analyses were conducted after passage 2, at 6 or moredays post infection. Data are presented as mean±s.e.m. *P<0.05.**P<0.01. ***P<0.001. Eight days post deletion, NSPCs exhibited a 73%reduction in NAD+ levels that was rescued by concurrent NMNadministration, and without being limited by theory, further supportingthe notion that Nampt activity is the predominant source of NSPCs NAD+levels (FIG. 18A). Neurospheres were isolated from Namptflox/flox miceand infected with a Cre-recombinase expressing adenovirus (Nampt AD-Cre)or a control adenovirus expressing LacZ (Nampt AD-LacZ).

Like FK866-treated cultures, proliferating Nampt Ad-Cre infected NSPCsdisplayed reduced cell number (FIG. 18B). Nampt Ad-Cre NSPCs were unableto increase their cell number between 24 and 144 hours of culture. Incontrast, Nampt AD-LacZ infected cells were able to exponentiallyincrease their cell number over 13-fold in this time frame. Consistentwith this finding, Nampt Ad-Cre infected NSPCs also showed a 49%reduction in diameter relative to Nampt AD-LacZ infected NSPCs,indicative of reduced proliferation (FIGS. 18C-D). Since NSPCself-renewal decisions can also contribute to cell number, the inventorsassessed secondary neurosphere formation, an assay that quantifies theability of neurosphere inhabitant cells to reformulate neurospheres upondissociation. Nampt Ad-Cre infected cells generated 63% fewer secondaryneurospheres than did Nampt AD-LacZ infected cells (FIG. 18E). NamptAD-LacZ and Nampt Ad-Cre NSPCs exhibited no difference in thepercentages of TUNEL- or activated caspase 3-positive cells as well asno difference in activated caspase3 immunoreactivity as detected byimmunoblotting, without being limited by theory, indicating that theobserved phenotypes upon loss of Nampt are not primarily due to celldeath (FIG. 25E-F). As a positive control for activated caspase 3immunoreactivity, indicated samples were treated with stauroporine (1mM) (n=6). To see if Nampt Ad-Cre infected neurospheres could bereactivated to proliferate, the inventors plated equal numbers of NamptAD-LacZ and Nampt Ad-Cre cells after the second passage and culturedthem in the presence or the absence of NMN. NMN treatment was able tofully reactivate the proliferative potential of Nampt Ad-Cre cells (FIG.18F-G). Collectively, without being limited by theory, these resultssuggest that Nampt-mediated NAD+ biosynthesis plays a role for NSPCs tosuccessfully progress through the cell cycle.

Whereas the inventors did not observe a difference in NSPC fatedecisions in the neurogenic environment of the SGZ in vivo, theinventors detected a decrease in self-renewal decisions. To see if thiswould occur in the absence of the influences of the SGZ niche, theinventors differentiated dissociated neurospheres and assessed theproportion of resulting cell types by immunofluorescence after 6 to 7days of differentiation induced by removal of growth factors (FIG. 18H,FIG. 25G). Differentiated Nampt Ad-Cre NSPCs exhibited a 90% reductionin oligodendrocytes (FIG. 18I). In contrast, Nampt Ad-Cre infected NSPCsexhibited no change in the generation of Gfap+ cells (FIG. 18J). Geneticknockdown of Nampt also significantly but more mildly decreased thegeneration of neurons (by 43% β-III-tubulin+, FIG. 18K). Thus, thedecrease in oligodendrocytes was not due to an increase in neuronalfate. As Gfap can recognize both NSPCs and mature astrocytes, theinventors employed Nestin and S100β to distinguish whether the decreasein oligodendrocytes we observed upon Nampt knockdown was due to a cellfate choice in these directions. While there was no detectable change inthe generation of S100β+ mature astrocytes in Nampt Ad-Cre cultures,there was a 4-fold increase in the percentage of Nestin+ cells (6% inNampt Ad-LacZ cells; 23% in Nampt Ad-Cre cells), without being limitedby theory, suggesting quiescence rather than precocious astrocyticdifferentiation (FIG. 18L). All of these effects were rescued bytreatment with NMN. The inventors also observed a mild increase inTUNEL+ cell death under these conditions (33% increase relative to NamptAd-LacZ cells). *, {circumflex over ( )}, and #indicate statisticalsignificance between Nampt AD-LacZ and Nampt AD-Cre, Nampt AD-LacZ andNampt AD-LacZ+ NMN, and Nampt AD-Cre and Nampt AD-Cre+ NMN,respectively. Data are presented as mean±s.e.m. *P<0.05. **P<0.01.***P<0.001. (FIGS. 18A-L) Together, without being limited by theory,these data suggest that genetic knockdown of Nampt prevents thesuccessful differentiation of oligodendrocytes from NSPCs, potentiallydue to quiescence as indicated by a retention of NSPC characteristics.

Example 13

This example illustrates that genetic knockdown of Nampt impairs OPCformation in vitro.

Since differentiation using a nonspecific lineage differentiationprotocol (by removal of growth factors) revealed a specific requirementfor Nampt in the successful generation of O4+ immature oligodendrocytes,the inventors next asked which stage(s) of NSPC differentiation intooligodendrocytes depends on Nampt by employing a differentiationprotocol that promotes the oligodendrocyte lineage (FIG. 19A, FIG. 26A).Neurospheres were isolated from Namptflox/flox mice and infected with aCre-recombinase expressing adenovirus (Nampt AD-Cre) or a controladenovirus expressing LacZ (Nampt AD-LacZ). As previously observed usinga nonspecific lineage differentiation protocol, the proportion of O4+intermediate oligodendrocytes was dramatically decreased in Nampt Ad-Crecultures at 6-7 days post differentiation (FIG. 19B). The inventorsobserved that ablation of Nampt resulted in a decreased pool of OPCs(Pdgfrα+), but an increased pool of Nestin+ NSPCs. The proportion ofGfap+ astrocytes/NSPCs also mildly increased. To investigate whether thedepletion of the OPC population was preexisting to or induced upondifferentiation, the inventors assessed the OPC population presentduring proliferation. Dissociated neurospheres were cultured inproliferation media containing PDGFαα. (FIG. 26B) and after 2 days ofdifferentiation (FIG. 19C), a time point that enriches for OPCs asassessed by immunofluorescence. Both of these time points also showedloss of OPCs (Pdgfrα+, Olig2+). These results support that, withoutbeing limited by theory, Nampt is plays a role for NSPCs todifferentiate into OPCs.

The inventors observed that Sirt2 was upregulated during oligodendrocytedifferentiation in vitro and expressed in the SGZ in Nampt+ cells andNestinGFP+ NSPCs (FIG. 26C). The inventors acutely treated NSPCs withthe selective inhibitor of Sirt2, AGK2, or the Sirt1 inhibitor, EX527.Whereas both inhibitors acutely suppressed oligodendrocyte formation(O4+, FIG. 19D), neither chronic ablation of Sirt2 in the NSPCs isolatedfrom Sirt2 mice nor Cre adenovirus mediated knockdown of Sirt1 inSirt1^(flox/flox) derived neurospheres affected oligodendrogenesis(Pdgfrα+, Olig2+, O4+), except that Sirt1 deficiency affected theproduction of O4+ intermediate oligodendrocytes (FIG. 19E). Theinventors generated Sirt1/Sirt2-double knockout (Sirt1/2 DKO)neurospheres. Consistent with the inhibitor studies (FIG. 19D),dissociated Sirt1/2 DKO neurospheres were unable to form oligodendrocytelineage cells upon differentiation (FIG. 19E). To assess the role ofSirt1 and Sirt2 downstream of Nampt activity, the inventors examined theexpression of genes associated with OPC formation in Nampt Ad-Creneurospheres, Sirt1/2 DKO neurospheres, and their respective controls.Dissociated Nampt Ad-Cre and Sirt1/2 DKO neurospheres showed similardecreases in the mRNA expression of Pdgfrα, Sox10, Nkx2.2 after 2 daysof differentiation (FIG. 19F-G). Dissociated Nampt Ad-Cre and Sirt1/2DKO neurospheres respectively exhibited similar increases in theexpression of p21 (cdkn1a). Olig1 expression showed no change or slightreduction by these genetic ablations, potentially due to its lesserexpression in NSPCs relative to Olig2 (Ligon et al., 2007) andpredominant roles in oligodendrocyte maturation and remyelination ratherthan specification. Neither Cre mediated knockdown of Sirt1 inneurospheres nor neurospheres cultured from whole-body Sirt1^(−/−) orSirt2^(−/−) mice exhibited defects in proliferation (FIG. 26G-J). Dataare presented as mean±s.e.m. *P<0.05. **P<0.01. ***P<0.001. Theinventors conclude that Sirt1 and Sirt2 can redundantly mediate NSPCdifferentiation into OPCs.

Example 14

This example illustrates adult NSPC-specific deletion of Nampt impairsNSPC differentiation in response to insult in vivo.

The inventors observed Nampt ablation on NSPC differentiation into OPCsin vitro, but not oligodendrogenesis in the SGZ of iNSPC-Nampt-KO micein vivo (FIG. 16I). The inventors assessed the percentage of NestinGFP+cells that expressed Olig2 in the SVZs of iNSPC-GFP and iNSPC-Nampt-KOmice (FIG. 20A). iNSPC-Nampt-KO mice showed a lower percentage ofoligodendrocytes generated from adult NSPCs.

The inventors employed the cuprizone model of demyelination andremyelination. Specifically, the inventors fed 6- to 9-week-oldiNSPC-Nampt-KO and littermate control mice (iNSPC-GFP) a diet containing0.2% cuprizone for 4-5 weeks, inducing deletion of Nampt in the adultNestin+ population the week before starting the cuprizone diet (FIG.20B) (Skripuletz et al., 2011). To ensure that analysis of progeny ofadult Nestin+ cells, all mice in our cohort expressed Cre recombinaseunder the inducible Nestin promoter (Nestin-CreERT2) (Lagace et al.,2007) and the aforementioned Cre recombinase responsive GFP reportertransgene. The analysis focus was on lineage tracer marked (NestinGFP+)cells.

Cuprizone feeding did not alter the total number of NestinGFP+ cellspresent in the iNSPC-GFP DG (FIG. 27A-C), suggesting, without beinglimited by theory, that NSPC proliferation was unaltered. Data arepresented as mean±s.e.m. *, {circumflex over ( )}<0.05. **, {circumflexover ( )}{circumflex over ( )}<0.01. ***, {circumflex over( )}{circumflex over ( )}{circumflex over ( )}P<0.001. Cuprizone fedmice exhibited an increased percentage of NestinGFP+ cells thatco-localized with the NSPC markers Nestin+(from 13 to 35%) andGfap+(from 19 to 41%), suggesting that, without being limited by theory,cuprizone treatment prevented SGZ NSPCs from terminally differentiatingand instead resulted in their retention of NSPC characteristics, whichcould occur through increased self-renewal decisions and/or quiescence(FIG. 27D-E). The inventors next assessed colocalization betweenNestinGFP and oligodendrocyte specific markers, Sox10 and APC. However,the SGZ did not substantially produce oligodendrocytes even in responseto demyelination (FIG. 27F-G Therefore, without being limited by theory,SGZ NSPCs do not appear to be the main mediators of short-termremyelination in the hippocampus.

The inventors assessed the fate decisions of migratory cells derivedfrom the adult Nestin+ population in the subcallosal zone of the corpuscallosum (FIG. 27A). In the iNSPC-GFP CC, virtually no NestinGFP+ orNestin+ cells were seen in regular chow fed mice (FIG. 20C-D, FIG. 27A).There were no differences in the number of NestinGFP+ cells in the CCbetween control and iNSPC-Nampt-KO mice, suggesting, without beinglimited by theory, that loss of Nampt neither affected insult-inducedNSPC proliferation or migration. In the iNSPC-GFP CC, cuprizone feedingsignificantly increased the percentage of NestinGFP+Nestin+ cells (from3 to 41%) but decreased the NestinGFP+Gfap+(from 60 to 24%) doublepositive cells, suggesting, without being limited by theory, increasedself-renewal fate decisions at the expense of astrocytic fate decisions(FIG. 20E-G). In control mice, cuprizone feeding also increased thenumber of NestinGFP+Sox10+(from 2 to 16%) and NestinGFP+Apc+(from 0 to4%) double positive cells, suggesting increased oligodendrocyte lineagefate decisions (FIG. 20H-1).

In contrast, the NestinGFP+ cells in the iNSPC-Nampt-KO CC showedsignificantly less colocalization with Nestin, Sox10, and Ape and morecolocalization with Gfap (FIG. 20F-1). Interestingly, Nampt was onlyexpressed in the CC upon insult (FIG. 20J, FIG. 27H). Moreover, Namptcolocalized with markers of NSPCs (Sox2, FIG. 27H) and oligodendrocytes(Olig2, FIG. 20J). These results suggest, without being limited bytheory, that Nampt is specifically expressed in SGZ/SVZ derivedremyelinating NSPCs and plays a role in oligodendrogenesis in responseto insult.

Example 15

This example illustrates a model for the role of Nampt-mediated NADbiosynthesis in NSPCs without being limited by theory.

Nampt-mediated NAD+ biosynthesis promotes NSPC self-renewal,proliferation and differentiation into oligodendrocytes. While themechanism by which Nampt promotes self-renewal and proliferation remainsunidentified, Nampt-mediated NAD+ biosynthesis activates Sirt1 and Sirt2to promote NSPC oligodendrocyte lineage fate decisions by a mechanisminvolving transcriptional downregulation of Pdgfrα, Sox10, and Nkx2.2and transcriptional upregulation of p21 (cdkn1a). Sirt1 and Sirt2 mayact via an effect on Olig2 activity. (FIG. 21)

The inventors showed that the NSPC pool decreased with age and thatlong-term NMN administration was able to maintain the NSPC pool. Theinventors assert that a higher dosage of NMN can be used to promote NSPCproliferation. Intraperitoneal injection of NMN substantially increaseshippocampal NAD+ levels within 15 minutes (FIG. 23G), without beinglimited by theory, suggests that NMN can cross the blood-brain barrier.

As E2F1-deficient mice have significantly reduced hippocampal NSPC death(Cooper-Kuhn et al, 2002), without being limited by theory, the observeddecrease in E2F1 upon inhibition of Nampt may explain this phenomenon(FIG. 17E). The inventors also observed that loss of Nampt activityspecifically downregulated Cyclin E and A expression. E-type cyclinsregulate G1 progression. The inventors observed downregulation of E2F1expression, which transcriptionally regulates Cyclin E, therefore,without being limited by theory, it is likely that the downregulation ofE2F1 contributes to the downregulation of Cyclin E. The inventors alsoobserved upregulation of p21 upon loss of Nampt. Thus, the upregulationof p21 that we see upon loss of Nampt, without being limited by theory,may also contribute to the downregulation of E2F/Cyclin E activity. AsCyclin A expression is induced after E2F and Cyclin E (Wong et al,2011), the changes in Cyclin A levels are likely downstream of both theaforementioned changes. While we have linked Nampt to the E2F/Cyclin Epathway, connecting mediator(s) remain unclear. The inventors foundneither Sirt1 nor Sirt2 to be downstream of the effect of Nampt-mediatedNAD+ biosynthesis on proliferation. While it is possible that Sirt1/2function redundantly to mediate NSPC proliferation, the relatively lowexpression of Sirt2 in NSPCs (FIG. 26G) makes this possibility unlikely,without being limited by theory.

The present inventors revealed that ablation of Nampt specificallyreduced the proportion of NSPC-generated Pdgfrα+ OPCs as well as thetranscription of Pdgfrα, Sox10, and Nkx2.2 but upregulated theexpression of p21. The results showed that in neurospheres, treatmentwith NMN rescued defects in oligodendrogenesis caused by a reduction inNAD+ levels. Furthermore, systemic NMN administration was able tosubstantially augment hippocampal NAD+ levels and increase the NSPCpool. Thus, NMN administration could be an efficient intervention toenhance the NSPC pool and promote remyelination by activating endogenousNSPCs during the aging process and/or in neurodegenerative diseases thatcause demyelination. The results provide evidence of the therapeuticpotential of Nampt-mediated NSPC self-renewal, proliferation, anddifferentiation into oligodendrocytes.

Example 16

This example illustrates an increase in bone density in aged individualsby NMN administration.

The inventors measured the bone mineral density (BMD) of control andNMN-treated mice at the 12-month time point of a 12 month long NMNadministration experiment by dual-energy X-ray absorptiometry (DXA). Atthis time point, mice were 17-18 month old. The inventors found thatNMN-treated mice showed increases in the BMD in a dose-dependent manner,and the difference between control and 300 mg/kg groups is statisticallysignificant (P=0.037, ANOVA, Tukey HSD post hoc test). Mice from 100 and300 mg/kg groups showed 2.8% and 5.9% increases in the BMD,respectively. Although age-associated BMD loss is extensively variedamong mouse strains (http://phenome.jax.org), the extent of theseobserved BMD increases is significant, indicating that NMN is able toenhance the BMD in aged individuals. These data indicate that NMNadministration can be used to treat age-associated osteoporosis inhumans.

Example 17

This example illustrates characterization of loss of NAMPT-mediated NADbiosynthesis on PR neuron survival.

The inventors examined the effect of selectively disrupting NADbiosynthesis within PR. The inventors utilized the cre-lox strategy togenerate mice that had NAMPT conditionally deleted from either rods(NAMPT rod-CKO) or cones (NAMPT cone-CKO). The NAMPT fl/fl mice as wellas the rhodopsin-cre and cone opsin-cre mice have been previouslycharacterized. Both rod and cone cko mice are generated with normalMendelian frequencies and are born normal with no observable systemicabnormalities (data not shown). All structural and functional analysesperformed in CKO mice are analyzed in comparison to littermate controls.Rods constitute a majority of the PR neurons (97% of allphotoreceptors). Retinas from NAMPT rod-CKO mice showed a significantreduction of NAMPT within rods by PCR, immunohistochemistry andimmunoblotting (FIG. 29A-B). Biomicroscopic examination of NAMPT rod-ckomice demonstrated a degenerative phenotype characterized by massiveatrophy of the neurosensory retina, vascular attenuation with pigmentmottling and atrophy of the underlying retinal pigment epithelium.Neurosensory retinal degeneration was associated with secondary atrophyand pallor of the optic nerve (FIG. 29C).

Example 18

This example illustrates electroretinography (ERG) performed to measurePR neuron and retinal function.

NAMPT rod-CKO mice demonstrated a dramatic reduction in scotopic(rod-associated) and photopic (cone-associated) responses compared tolittermate control animals (FIG. 29D-H) Photopic visual acuitymeasurements confirmed vision loss in rod-CKO mice (FIG. 29G).Histopathologic examination of eyes from NAMPT rod-CKO mice wascharacterized by retinal degeneration with progressive loss of the outernuclear layer over time with significant reduction of retinal thicknessand subsequent extension of the neurodegeneration to multiple retinallayers (FIG. 29H). Normalized NAD measurements obtained from NAMPTrod-cko whole retinas showed a significant reduction in NAD which isespecially important given that NAMPT function is selectively eliminatedonly from rod PR neurons with other retinal cells being normal (FIG.29I). These results suggest that, without being limited to theory, ifenzymatic activity of NAMPT in NAD biosynthesis in rod PR neurons isnecessary for cell survival, intracellular conversion of NAM to NMN byNAMPT can play a role.

The present inventors determined that exogenous supplementation with NMNis able to rescue PR neurons from cell death in CKO mice.Intraperitoneal (i.p.) delivery was chosen to obtain early and sustainedlevels of NMN. In these experiments, NAMPT rod-CKO mice were given NMN(150 mg/kg) or PBS i.p. daily starting at day P5. ERG at 4 weeks in CKOmice treated with NMN showed significant rescue of photopic and scotopicfunction compared to PBS treatment (FIG. 29J-L). There was no effect ofNMN on littermate control animals.

Example 19

This example illustrates NMN rescue in NAMPT cone-CKO mice.

In these experiments, NAMPT cone-CKO mice (without NMN treatment)demonstrated similar but milder changes on biomicroscopy consistent withneuroretinal degeneration as seen in the rod-CKO mice (FIG. 30A). ERGdemonstrated significant and progressive decline in cone function asevidenced by reduced photopic responses over time with secondaryreduction in scotopic responses (FIG. 30B-D). These quantifiablestructural and functional changes were associated with decrease invisual acuity in cone-CKO mice (FIG. 30E). Histopathologic analysesconfirmed outer nuclear layer degeneration with subsequent multilayerretinal degeneration and cell death in cone-CKO mice similar to thechanges seen above for rod-CKO mice. As with NAMPT rod-CKO mice,delivery of NMN i.p. to NAMPT cone-CKO mice was also able to improve ERGfunction compared to PBS treated cone-CKO mice (FIG. 30 F-H). NMNtreatment had no effect on littermate controls (FIG. 33A-B). These datasuggest that, without being limited by theory, NAMPT-mediated NADbiosynthesis is necessary for the survival and function of both rod andcone PR neurons. Furthermore, providing NMN treatment is able to rescuePR neurons and vision.

The inventors used a 661W cone PR cell line and treated the cells withthe specific pharmacological NAMPT inhibitor FK866 (200 nM). FK866treatment of cone cells in vitro causes decrease in intracellular NADlevels and significant cell death after the 4 hours of treatment (FIGS.31I, 31J). Cell death progresses dramatically over the next 20 hours.NMN (100 μM) was able to completely rescue cells from death associatedwith FK866 treatment and restore NAD to normal levels (FIGS. 31I, 31K).These in vitro results confirm that NMN administration can promote PRneuron survival.

Example 20

This example illustrates that NAD-regulated PR survival is independentof individual sirtuins.

The inventors examined the effect of deletion of several sirtuins on PRsurvival. Sirt 1 PR CKO mice and Sirt 2-5−/− mice had normal retinal andPR neuron structure and function when examined by fundus biomicroscopyand ERG (FIG. 3a-j and FIG. S2). Sirt6−/− mice have a profoundneurodegenerative phenotype and die around 3-4 weeks of age. As such, weexamined sirt6 rod and cone conditional knockout mice that also hadnormal retinal structure and function (FIG. 31A-N, and FIG. 34A-F).Without being limited by theory, these findings demonstrate thatindividual sirtuins are not causative of NAMPT-mediated PR degeneration.

Example 21

This example illustrates that photoreceptor loss and blindness isassociated with mitochondrial dysfunction.

Electron microscopic examination demonstrated dysmorphic changes in theretinal inner segments along with disruption of the outer segments inrod CKO mice but showed normal cellular organization and sub-cellularstructures in littermate controls at 4 weeks of age (FIG. 32A-C, 32D).By 4 weeks of age, the mitochondrial numbers in CKO retinas weresignificantly reduced, the mitochondria were rounded and constrictedwith loss of cristae as opposed to the normally elongated mitochondriawith healthy cristae seen in age-matched littermate control mice (FIG.32A-D). There was an abundance of degenerative vacuoles with ingestedorganelles including mitochondria in rod-CKO mice with no suchstructures identified in littermate wild type controls. At 3 weeks ofage, subtle changes in inner segments could be identified in rod-CKOmice although they were not as dramatic as those seen by 4 weeks (FIG.35). These results suggest that, without being limited by theory, NADdeficiency might impair mitochondrial structure and function. Theinventors treated 661W cone cells with NAMPT inhibitor FK866 (200 nM).In the oxygen consumption rate measurement assay, multiple aspects ofmitochondrial function were analyzed. As shown in FIG. 32E, maximalrespiration was significantly reduced in 661W cone cells afterinhibition of NAMPT function (FIG. 32E). NMN treatment was able tocompletely reverse the effects of FK866 on cone cell photoreceptormitochondrial function confirming the role of NAD in NAMPT-mediatedeffects on PR neurons (FIG. 32E).

A non-biased metabolomic analysis using mass spectrophotometry (GC-MSand LC-MS) was performed on retinas isolated from NAMPT rod CKO mice andcompared to littermate control retinas. Significant differences wereidentified in mitochondrial metabolites involved in the TCA cycle.

REFERENCES

-   Amrnett, H. A., et al., Science 306, 2111-2115, 2004.-   Artegiani, B., et al. Aging 4, 176-186 2012.-   Ben Abdallah, N. M., et al. Neurobiol. Aging 31, 151-161, 2010.-   Bieganowski, P., et al., Cell 117, 495-502, 2004.-   Bouab, M., et al. Neuroscience 173, 135-149, 2011.-   Bundgaard, H., ed., Design of Prodrugs, Elsevier, 1985.-   Bundgaard, H., Advanced Drug Delivery Reviews 8, 1-38, 1992.-   Canto, C., et al., Cell-Metab. 15, 838-847, 2012.-   Carlson, L. A., J. Intern. Med. 258, 94-114, 2005.-   Cheadle, C., et al. J. Mol. Diagn. 5, 73-81, 2003.-   Chen, J., et al., Hepatology 57, 2287-2298, 2013-   Colak, D., et al., J. Neurosci. 28, 434-446, 2008.-   Collins, P. B., et al., Biochem. J. 125, 117P, 1971.-   Collins, P. B., et al., J. Biol. Chem. 247, 778-783, 1972.-   Cooper-Kuhn, C. M., et al., Mol. Cellular Neurosci. 21, 312-323,    2002.-   Dasgupta, B., et al., J. Neurosci. 25, 5584-5594, 2005.-   Decker, L., et al., Neurosci. Res. 69, 763-771, 2002-   Deng, W., et al., Nature Reviews 11, 339-350, 2010.-   Dienel, G. A., et al., Neurochem. Int'l. 48, 586-595, 2006.-   Di Girolamo, M., et al., FEBS J. 272, 4565-4575, 2005.-   Doucette, J. R., et al., Cell. Molec. Neurobiol. 30, 607-629, 2010.-   Encinas, J. M., et al., Cell Stem Cell 8, 566-579, 2011-   Folmes, C. D., et al., Cell Stem Cell 11, 596-606, 2012.-   Franklin, R. J., et al., Nature Reviews 9, 839-855, 2008.-   Friebe, D., et al., PloS One 6, c19526, 2011.-   Fu, J., et al., Diabetologia 49, 1027-1038, 2006.-   Gao, Q., et al., Int. J. Dev. Neurosci. 25, 349-357, 2007.-   Garten, A., et al., Trends Endocrinol. Metab. 20, 130-138, 2009.-   Gudi, V., et al., Brain Res. 1283, 127-138, 2009.-   Guillemin, G. J., et al., J. Neurosci. 27, 12884-12892, 2007.-   Hack, M. A., et al., Nature Neurosci. 8, 865-872, 2005.-   Hack, M. A., et al., Molec. Cell. Neurosci. 25, 664-678, 2004.-   Hara, N., et al., J. Biol. Chem. 282, 24574-24582, 2007.-   Hasmann, M., et al., Cancer Res. 63, 7436-7442, 2003.-   Higuchi, T. and Stella, V., Pro-drugs as Novel Drug Delivery    Systems, A.C.S. Symposium Series 14, 1975.-   Hisahara, S., et al., Proc. Nat'l. Acad. Sci. USA 105, 15599-15604,    2008.-   Houtkooper, R. H., et al., Endocr. Rev. 31, 194-223, 2010.-   Husain, J., et al., Brain Res. 698, 86-94, 1995-   Imai, S., FEBS Lett. 585, 1657-1662, 2011.-   Imai, S. et al., Trends Pharmacol. Sci. 31, 212-220, 2010.-   Imai, S., Curr. Pharm. Des. 15, 20-28 2009.-   Imai, S., Pharmacol. Res. 62, 42-47, 2010.-   Ito, K., et al. Nature Med. 18, 1350-1358, 2012.-   Jablonska, B., et al., Nature Neurosci. 13, 541-550, 2010.-   Jackson, E. L., et al., Neuron 5, 187-199, 2006.-   Jadasz, J. J., et al., Cell Tissue Res. 349, 331-347, 2012.-   Ji, S., et al., J. Mol. Cell Biol. 3, 351-359, 2011.-   Jin, K., et al., Aging Cell 2, 175-183, 2003.-   Jin, Y. H., et al., Biochem. Biophys. Res. Comm. 368, 690-695, 2008.-   Kakeya, N., et al., Chem. Pharm. Bull. 32, 692-698, 1984.-   Kim, H. S., et al., Cancer Cell 20, 487-499, 2011.-   Knobloch, M., et al., Nature 493, 226-230, 2013.-   Krogsgaard-Larsen, H. and Bundgaard, H., A Textbook of Drug Design    and Development, Chapter 5; “Design and Applications of Prodrugs”    113-191, 1991.-   Lagace, D. C., et al., J. Neurosci. 27, 12623-12629, 2007.-   Lau, C., et al., J. Biol. Chem. 285, 18868-18876, 2010.-   Lee, H. C., Sci. China Life Sci. 54, 699-711, 2011.-   Li, W., et al., J. Neurosci. 27, 2606-2616, 2007.-   Ligon, K. L., et al., Neuron 53, 503-517, 2007.-   Liu, A., et al., EMBO J. 25, 4833-4842, 2006-   Lu, P. P., et al., J. Neurosci. 32, 8012-8023, 2012.-   Lu, Q. R., et al., Cell 109, 75-86, 2002.-   Lugert, S., et al., Cell Stem Cell 6, 445-456, 2010.-   Luo, J., et al., Cell 107, 137-148, 2001.-   Madisen, L., et al., Nature Neurosci. 13, 133-140, 2010.-   Mehta, S., et al., Cancer Cell 19, 359-371, 2011.-   Menn, B., et al., J. Neurosci. 26, 7907-7918, 2006.-   Nait-Oumesmar, B., et al., Euro. J. Neurosci. 11, 4357-4366, 1999.-   Nielsenw, N. M, and Bundgaard, H., J. Pharm. Sci. 77, 285-298, 2006.-   Norkute, A., et al., J. Neurosci. Res. 87, 1343-1355, 2009-   Outeiro, T. F., et al., Science 317, 516-519, 2007.-   Peck, B., et al., Molec. Cancer Therap. 9, 844-855, 2010.-   Picard-Riera, N., et al., Proc. Nat'l. Acad. Sci. USA 99,    13211-13216, 2002.-   Plassman, B. L., et al., Ann. Internal Med. 148, 427-434, 2008.-   Polager, S., et al., Nat. Rev. Cancer 9, 738-748, 2009.-   Prozorovski, T., et al., Nature Cell Biol. 10, 385-394, 2008.-   Rafalski, V. A., et al., Nature Cell Biol. 15, 614-624, 2013.-   Revollo, J. R., et al., J. Biol. Chem. 279, 50754-50763, 2004.-   Revollo, J. R., et al., Cell Metabolism 6, 363-375, 2007.-   Rongvaux, A, et al., J. Immunol. 181, 4685-4695, 2008.-   Roche, E. B., Bioreversible Carriers in Drug Design: Theory and    Application, Elmsford, N.Y.: Pergamon Press 1987.-   Rothgiesser, K. M., et al., J. Cell Sci. 123, 4251-4258, 2010.-   Saharan, S., et al., J. Neurosci. Res. 91, 642-659, 2013.-   Saito, K., et al., Proc. Nat'l. Acad. Sci. USA 106, 8350-8355, 2009.-   Sanchez-Abarca, L. I., et al., Glia 36, 321-329, 2001.-   Sassone-Corsi, P., Endocrinol. 153, 1-5, 2012.-   Schreiber, V., et al., Nat. Rev. Mol. Cell Biol. 7, 517-528., 2006.-   Sim, F. J., et al., J. Neurosci. 22, 2451-2459, 2002.-   Skripuletz, T., et al., Histol. Histopath. 26, 1585-1597, 2011.-   Soundarapandian, M. M., et al., (2011) Scientific Reports 1, 2, 2011-   Stein, L. R., et al., Trends Endocrin. Metabol. 23, 420-428, 2012.-   Steiner, B., et al., Glia 46, 41-52, 2004.-   Stoll, E. A., et al., J. Biol. Chem. 286, 38592-38601, 2011.-   Sun, Y., et al., Neuron 69, 906-917, 2011.-   Takebayashi, H., et al., Curr. Biol. 12, 1157-1163, 2002.-   Tyler, W. A., et al., Glia 59, 1754-1769, 2011.-   Van Leeuwen, I. M., et al., Molec. Cancer Ther. 12, 471-480, 2013.-   Verderio, C., et al., J. Neurochem. 78, 646-657, 2001.-   Voloboueva, L. A., et al., J. Neurosci. 30, 12242-12251, 2010.-   von Bohlen und Halbach O., Cell Tissue Res. 345, 1-19, 2011.-   Wang, C., et al., Nature Cell Biol 8, 1025-1031, 2006.-   Wang, P., et al., Ann. Neurol. 69, 360-374, 2011.-   Wang, W., et al., J. Neurosci. 31, 9746-9751, 2011.-   Wegner, M., et at, J. Mol. Neurosci. 35, 3-12, 2008.-   Wilhelm, F., et al., J. Neurosci. Res. 89, 1956-1964, 2011.-   Wong, J. V., et al., Cell Cycle 10, 3086-3094, 2011.-   Yang, H., et al., Cell 130, 1095-1107, 2007.-   Yoshino, J., et al., Cell Metabol. 14, 528-536, 2011.-   Zhang, J., et al., Cell Stem Cell 11, 589-595, 2012.-   Zhang, W., et al., J. Cereb. Blood Flow Metab. 30, 1962-1971, 2010.-   Zhang, Y., et al., Biochem. Biophys. Res. Comm. 404, 610-614, 2011.-   Zhou, Q., et al., Cell 109, 61-73, 2002.

All publications cited in this application are herein incorporated byreference in their entirety as if each individual publication, patent,patent application or other reference were specifically and individuallyindicated to be incorporated by reference. Applicant reserves the rightto challenge the accuracy and pertinency of the cited references.

What is claimed is:
 1. A method of treating dry eye in a subject,comprising: administering to a subject in need of treatment apharmaceutically effective amount of nicotinamide mononucleotide (NMN).2. A method in accordance with claim 1, wherein the dry eye isage-associated dry eye.
 3. A method in accordance with claim 1, whereinthe NMN is administered orally in a pharmaceutically acceptableformulation selected from the group consisting of a pill, a tablet, acaplet, a capsule, a chewable tablet, a quick dissolve tablet, a powder,a granule, an effervescent tablet, a hard gelatin capsule, a softgelatin capsule, a non-aqueous liquid, an aqueous liquid, a suspension,a solution, an emulsion, a syrup, a sterilized aqueous suspension, asterilized aqueous solution, a non-aqueous suspension, a non-aqueoussolution, and a lyophilized formulation.
 4. A method in accordance withclaim 1, wherein the NMN is administered parenterally in apharmaceutically acceptable formulation selected from the groupconsisting of a non-aqueous liquid, an aqueous liquid, a suspension, asolution, an emulsion, a syrup, a sterilized aqueous suspension, asterilized aqueous solution, a non-aqueous suspension, a non-aqueoussolution, a lyophilized formulation.
 5. A method in accordance withclaim 1, wherein the NMN is administered intravenously.
 6. A method inaccordance with claim 1, wherein the NMN is administered enterically. 7.A method in accordance with claim 1, wherein the NMN is administeredparenterally.
 8. A method in accordance with claim 1, wherein the NMN isadministered is topically.
 9. A method in accordance with claim 8,wherein the topical administration is administering an eye drop.
 10. Amethod in accordance with claim 1, wherein the NMN is administeredintraocularly.
 11. A method in accordance with claim 1, wherein thesubject is a mammal.
 12. A method in accordance with claim 1, whereinthe subject is a human.
 13. A method of increasing tear production in asubject, comprising: administering to a subject in need of treatment apharmaceutically effective amount of nicotinamide mononucleotide (NMN).14. A method in accordance with claim 13, wherein the NMN isadministered orally in a pharmaceutically acceptable formulationselected from the group consisting of a pill, a tablet, a caplet, acapsule, a chewable tablet, a quick dissolve tablet, a powder, agranule, an effervescent tablet, a hard gelatin capsule, a soft gelatincapsule, a non-aqueous liquid, an aqueous liquid, a suspension, asolution, an emulsion, a syrup, a sterilized aqueous suspension, asterilized aqueous solution, a non-aqueous suspension, a non-aqueoussolution, and a lyophilized formulation.
 15. A method in accordance withclaim 13, wherein the NMN is administered parenterally in apharmaceutically acceptable formulation selected from the groupconsisting of a nonaqueous liquid, an aqueous liquid, a suspension, asolution, an emulsion, a syrup, a sterilized aqueous suspension, asterilized aqueous solution, a non-aqueous suspension, a non-aqueoussolution, a lyophilized formulation.
 16. A method in accordance withclaim 13, wherein the subject is a mammal.
 17. A method in accordancewith claim 13, wherein the subject is a human.
 18. A method inaccordance with claim 13, wherein the NMN is administered is topically.19. A method in accordance with claim 18, wherein the topicaladministration is administering an eye drop.
 20. A method in accordancewith claim 13, wherein the NMN is administered intraocularly.
 21. Amethod of suppressing an age-associated accumulation of intraretinaldeposits in a subject, comprising: administering to a subject in need oftreatment a pharmaceutically effective amount of nicotinamidemononucleotide (NMN).